Progress report on hair keratin research.

Evidence is given for the progress in hair keratin research by bringing out four examples from the recent hair science literature. 1 Kon et al. [1] published a new method of solubilization and fractionation of the matrix and the microfibril proteins and found a significant decrease in the 'intact' microfibril keratin at the tip end of permed hair. 2 Contrary to the previous view of the existence of four major and one minor hair keratin pair, Langbein et al. [2, 3] showed that there are nine type I hair keratins and six type II hair keratins and drew up a two-dimensional catalogue of human hair keratins. 3 To collectively describe the extremely complex expression pattern of human type I and type II hair keratins in the hair follicle, Langbein et al. [2, 3] have summarized the corresponding RNA expression profiles of the various hair keratins schematically. 4 Contrary to the previous assignment of keratins exclusively to the microfibrils in the cortex, the mRNA expression studies of Langbein et al. [2, 3] implied that any hair cuticle cell leaving the living cell compartment contains four different hair keratins.

My journey from wool research to insulin.

This paper is an autobiographical study of the author's early work on the chemical cross-linking of proteins as well as on oligomer and peptide synthesis from 1949 in Heidelberg until the synthesis of insulin in 1963 in Aachen.

The preoperative prevalence of deep vein thrombosis in patients with femoral neck fractures and delayed operation.

Out of 61 consecutive patients admitted for femoral neck fracture 21 patients had a delay to operation for more than 48 h from the time of injury. We studied these patients prospectively for the presence of deep-vein thrombosis (DVT). 13 (62%) had venographic evidence of thrombosis. All occurred in the broken limb. Five patients had bilateral thrombosis. The delay alone seems to be the major risk factor for thrombosis irrespective of age, fracture type, premorbid mobility and coexisting illness. The prevalence of preoperative DVT 48 h after injury approaches the reported postoperative incidence of DVT, which suggests that DVT will occur in a high proportion of patients regardless of treatment and prophylaxis. We recommend that those patients, in whom operation is delayed, should be routinely investigated for the presence of thrombosis preoperatively and a prophylactic vena cava filter should be considered when major deep vein thrombosis occurred.

Hair sulfur amino acid analysis.

This overview emphasizes present aspects of sulfur-containing amino acids in hair. A selection of analytical procedures to determine cystine, cysteine, S-sulfocysteine, cystine oxide, cysteic acid, lanthionine and lysinoalanine are presented. The methods relate to intact hair or partial and total hydrolysates and comprise chromatography, titration, colorimetry, polarography and spectroscopy. For the analysis of cysteine, cystine and cystine oxides, polarography and spectroscopy are the methods of choice. Cysteic acid, lanthionine and lysinoalanine are analysed by means of ion-exchange chromatography (Spackman et al., 1958) after total hydrolysis.

Structure-function relationships of des-(B26-B30)-insulin.

In order to study the role of the amino acid in position B25 and its environment in shortened insulins, a series of analogues was prepared with the following modifications: 1, Stepwise shortening of the B-chain including replacements of TyrB26 and ThrB27 by glycine; 2, substitutions at the carboxamide nitrogen of des-(B26-B30)-insulin-B25-amide by apolar, polar or charged residues of various chain lengths; 3, replacement of PheB25 by asparagine-amide, phenylalaninol or a series of alkyl and aralkyl residues. Trypsin-catalyzed semisyntheses were performed with Boc-protected or unprotected des-octapeptide-(B23-B30)-insulin and synthetic peptides. Relative receptor binding and in vitro bioactivity of [AsnB25]-des-(B26-B30)-insulin-B25-amide was 227 and 292% (on insulin), other activities ranged between 1 and ca. 200%. We make the following conclusions. An L-amino acid is essential in position B25. The B25-carbonyl and NH groups favour high binding and "superpotency", but are not indispensible for receptor contacts. For high affinity receptor interaction, the planarity at the C gamma-atom and the distance of B25-side-chain branching in position B25 are important, but an aromatic ring is not necessary.

Use of Z-amino acid-glyceryl esters in protease catalyzed peptide synthesis.

The alpha-glyceryl esters of Z-Gly, Z-Phe and Z-Tyr were synthesized and their use for protease catalyzed peptide synthesis was studied. Three enzymes isolated from crude papain were compared in their catalytic potency. Syntheses with alpha-chymotrypsin were performed in a biphasic system.

Hormone binding site of the insulin receptor: analysis using photoaffinity-mediated avidin complexing.

A trifunctional reagent was designed which allows derivatization of ligands, particularly peptides and proteins, for subsequent photoaffinity labelling of receptors and specific isolation of the covalent complex or its fragments. B29-(2-nitro-4-azidophenyl)-biocytinyl-insulin (NB-insulin) was synthesized, radioiodinated, and the B26-mono-iodo derivative isolated by HPLC. It was used to photoaffinity label human placental membranes and the purified insulin receptor. Extensive digestion of the covalent insulin-receptor complex with trypsin (EC 3.4.21.4) led to the generation of a fragment of Mr 14,000. Specific complexing with avidin, derivatized avidin or streptavidin could be demonstrated for the photoaffinity labelled alpha-subunit and the 14,000 core fragment. The latter was isolated (approx. 100 pmol from 3-4 placentae) by streptavidin affinity chromatography and HPLC. According to microsequencing based on the known primary structure of the insulin receptor, the N-terminus of the core peptide appears to be Leu20-His21-Glu22-Leu23. We thus conclude: a part of the insulin-binding region of the receptor is located close to the N-terminus of its alpha-subunit in a remarkably stable domain of the sequence 20--(approx.) 120.

Peptide analogues of the anaphylatoxin C3a; syntheses and properties.

The chemical syntheses of C-terminally shortened analogues of C3a, which is the best investigated anaphylatoxin and derives from the third component of complement system, is reported. The peptide assembly was performed with the solid-phase technique using a polyamide support and an orthogonal protection strategy. The base-labile Fmoc group was chosen for N alpha protection in combination with acid-labile side-chain protection. Excellent acylation yields could be obtained using HBTU (O-benzotriazolyl-N,N,N',N'-tetramethyluronium hexafluorophosphate) as activating reagent. With this methodology we synthesized eighteen different peptides with the following modifications: Varying the peptide length by sequential addition of glycine or arginine residues, prolongating the N-terminus with the Fmoc- or Fmoc-aminohexanoyl residues and exchanging the glycine in position 74 for alanine or D-alanine. We obtained two C3a analogues, Fmoc-YRAAALALAR and Fmoc-Ahx-YRRGRAAALGLAR, which were shown to be substantially more active than native C3a in the guinea-pig-platelet assay.

Contributions to the chemistry of human hair: II. Lipid chemical aspects of permanently waved hair.

Synopsis The cell membrane complex (CMC) and the internal lipids of untreated and permanent waved hair were investigated. The permanent waving procedure results in a lower amount of the cell membrane fraction that could be isolated compared to untreated hair. There is no difference in the amino acid composition of the CMC fractions. The internal hair lipids were extracted from the CMC fraction with chloroform/methanol. For untreated human hair the cell membrane complex yields 57% of lipid material, whereas from the CMC of permanent wave hair only 35% of lipids could be extracted. The polar lipids and the fatty acids are the main components of the internal hair lipids. After a permanent waving treatment there is a distinct decrease in the polar lipid fraction which is higher at alkaline (pH 9) than at neutral pH (pH 7). The reduction treatment at pH 9.0 also causes a cleavage of the fatty acid fraction in the internal hair lipids.

Contributions to the chemistry of human hair: III. Protein chemical aspects of permanent waving treatments.

Synopsis The influence of permanent waving on hair proteins was studied in order to obtain additional information about the chemistry of this cosmetic treatment. It was shown by amino acid analysis that with increasing reduction time during treatment fewer disulphide bonds were reformed in hair during subsequent reoxidation. Simultaneously, an increasing amount of sulphur-containing material is liberated from the hair, as demonstrated by the sulphur balance calculated from the sulphur-containing amino acids. The amount liberated is increased when an extensive soaking of the samples in water between the reduction and reoxidation step is performed. Comparing treatments with the use of reducing solutions of pH values between 7.5 and 10.0, it was found that the largest amount of cystine cleavage occurs at pH 9.0. All hair samples reduced at pH values above pH 8.5 showed incomplete reformation of the disulphide bonds during subsequent reoxidation. This was indicated by the content of free SH-groups and cysteic acid, as quantified by amino acid analysis. The damage to the hair proteins due to permanent waving was further confirmed by the determination of the pronase solubility. The reductive treatment of hair at pH 7.5 led to a relatively low degree of reduction, however all sulphur bonds were reformed during subsequent reoxidation.

Efficient synthesis of N alpha-acetylarginine methylamide.

A simple preparation of Ac-Arg-(p-TosH or HCl)-NHMe is described. The NG-protonated Z-Arg was coupled with methylamine by the mixed anhydride method. Z-Arg-NHMe was purified as a NG-p-toluene sulfonate salt by crystallization from water. After removal of the Z group by catalytic hydrogenation and acetylation Ac-Arg(p-TosH)-NHMe was obtained. Ac-Arg(HCl)-NHMe was prepared by chromatography of the NG-TosH derivative on Dowex 44 (in Cl- form).

Shortened insulin with enhanced in vitro potency.

After it has been shown that removal of residues B26-B30 leaves insulin with full biological activity, provided the new C-terminus is amidated (Fischer et al. (1985) Biol. Chem. Hoppe-Seyler 366, 521-525), it is demonstrated here that it does not even preclude enhancement of potency. 7 analogues of des-(B26-B30)-insulin-B25-amide were prepared by trypsin-mediated semisynthesis, the replacements being D-PheB24; HisB25, D-PheB25, TrpB25, TyrB25; D-PheB24,B25 and D-PheB24, TyrB25. Mere conversion of the configuration of B25-phenylalanine reduces in vitro potency to 0.5%. If B25-phenylalanine is, however, substituted by histidine or tyrosine activity is increased to 310 or 230, respectively. According to the features common to these two side chains, the favourable effect should be due to their ring structure with balanced aromatic and polar or H-bonding properties, respectively. The results indicate that in the complete insulin molecule the C-terminal pentapeptide modulates the subtle role that residues B24 and/or B25 play in receptor binding and activity; its presence may have a positive or negative effect. The drastic differences in activity between the shortened analogues are in no ways reflected in the CD spectra which are very similar, though clearly different from that of native insulin.

A study of the interaction of sodium dodecyl sulphate with the proteins of human heel stratum corneum.

Synopsis The aim of this research is to study the in vitro interaction of the anionic surfactant sodium dodecyl sulphate with human heel callus with special regard to the callus proteins. The sorption of SDS by callus is analysed at different pH values, demonstrating the expected maximum binding of the anionic surfactant at low pH. In both surfactant and blank solutions, swelling of the callus pieces occurs but to different extents. The sorption of SDS by callus is accompanied by a loss of free amino acids and proteins. The protein composition of the callus residues after chemical treatment and of the corresponding treatment baths is examined by amino acid analysis and shows differences in the respective molar amino acid ratios. Results obtained with more specific techniques, such as gel electrophoresis and immunoblotting, demonstrate identical as well as different protein components in the treatment baths depending on the experimental conditions (pH, blank or SDS). Although effects due to the surfactant treatment are in principle more distinct than with blank experiments, those of the latter cannot be neglected.

Contributions to the chemistry of human hair. 1. Analyses of cystine, cysteine and cystine oxides in untreated human hair.

Synopsis Amino acid analysis, photometry and polarography were selected as analytical methods for the determination of thiol and disulphide groups in untreated human hair and the results are discussed. The pre-treatments necessary for the different analytical methods, e.g., hydrolysis, to some extent already induce chemical changes of the cysteine and cystine derivatives leading to method-dependent differences in the results. In many cases the partial oxidation products of cystine are supposed to be responsible for this effect. Electron Spectroscopy for Chemical Analysis (ESCA), as an analytical method for the determination of the cystine oxides, was found to be inapplicable due to insufficient resolution and sensitivity, whereas by the use of Fourier Transform Infrared (FTIR) Spectroscopy the sulphur-oxygen vibrations could be analysed and cystine monoxide and cystine dioxide were detected.

Structure-function relationships of shortened [LeuB25]insulins, semisynthetic analogues of a mutant human insulin.

Replacement of B25-phenylalanine by leucine in the insulin sequence causes marked inactivation. The effect of this sequence variation was studied here in des-(B26-30)-insulin. [LeuB25]des-(B26-30)-insulin and its B25-amide were prepared by trypsin-mediated semisynthesis from N-terminally protected des-(B23-30)-insulin and synthetic tripeptides. The relative lipogenic potency in isolated rat adipocytes was 8.0% for the truncated analogue with a free B25-carboxyl function, and 18.1% for the amidated analogue. Binding to cultured human IM-9 lymphocytes was 4% and 9%, respectively. Thus, both shortened insulins are markedly more active than [LeuB25]insulin. The PheB25----LeuB25 substitution in both the shortened and the full sequence has a moderate effect on the CD spectrum, indicating that the gross main chain conformation is largely retained in both molecules. Independent of the substitution an absolute increase of the circular dichroism is observed upon amidation of the B25-carboxyl group.

The synthesis and some biological properties of N-(6-purinyl)peptides.

The chemical synthesis and biological properties of N-(6-purinyl)peptides are described. N-(6-Purinyl)amino-acid derivatives were synthesized and condensed with amino acid esters and peptide esters using the dicyclohexylcarbodiimide/N-hydroxysuccinimide method. The products were isolated via gel filtration on Sephadex G-10 in 0.05M NH4HCO3 followed by either ion exchange chromatography on SP-Sephadex or by preparative HPLC. The methyl esters were saponified and the tert-butyl ester group was removed by treatment with trifluoroacetic acid without damaging the purinyl residue. N-(6-Purinyl)peptides were characterised by chromatographic and spectroscopic methods. Acid hydrolysis of N-(6-purinyl)-L-amino acids caused the racemization of the neighbouring L-amino acid. Model studies were performed with N-(6-purinyl)-L-alanine, N-(6-purinyl)-D-alanine, N-(6-purinyl)-L-alanyl-L-leucine and N-(6-purinyl)-D-alanyl-L-leucine. After acid hydrolysis the N-(6-purinyl)amino acids were totally racemized and the N-(6-purinyl)dipeptides formed 14% of the enantiomer of alanine. The N-(6-purinyl)-omega-amino acids and the N-(6-purinyl)peptides were screened in a limited number of tests as immunomodulators (antibody-secretion, phagocytosis, cytostatic activity of macrophages) and as cytotoxic agents.

A shortened insulin with full in vitro potency.

Des[(B26-30)-pentapeptide]insulin-B25-amide was prepared from protected des-[(B23-30)-octapeptide]insulin (pig) and H-Gly-Phe-Phe-NH2 by trypsin-mediated semisynthesis in a yield of 9% (based on insulin). The analogue was characterized with respect to chemistry, biological function and CD spectroscopy. While des[(B26-30)-pentapeptide]insulin with free carboxylate group exhibited a typical insulin activity of only 25% in vitro, des[(B26-30)-pentapeptide]insulinamide was fully active. Therefore des[(B26-30)-pentapeptide]insulin meets all structural and dynamic requirements for recognition and binding of the receptor as well as exertion of the biological effect, provided that the negative charge in the hydrophobic environment of PheB25 is neutralized.

Immunostimulating hexapeptide from human casein: amino acid sequence, synthesis and biological properties.

A hexapeptide obtained from human casein by enzymatic digestion has been purified, sequenced and synthesized; its structure is: Val-Glu-Pro-Ile-Pro-Tyr. In vitro this hexapeptide stimulates the phagocytosis of opsonized sheep red blood cells by murine peritoneal macrophages. Administered intravenously to adult mice, it enhances the resistance to infection with Klebsiella pneumoniae.