[The "second life" of rifampicin]. / La deuxième vie de ta rifampicine.

Despite it was initially used and was restricted mainly for the treatment of tuberculosis, rifampicin gained in past years interest for the treatment of infections associated with prosthetic devices. Rifampicin has an excellent tissue penetration and a unique activity on bacteria in biofilms growing on the surface of prosthetic devices. The rapid emergence of resistance in bacteria constitutes the Achilles' heel and therefore rifampicin should be used with caution. This review addresses the mode of action of rifampicin and describes possible pitfalls of its use.

Effects of four-week high-fructose diet on gene expression in skeletal muscle of healthy men.

AIMS: A high-fructose diet (HFrD) may play a role in the obesity and metabolic disorders epidemic. In rodents, HFrD leads to insulin resistance and ectopic lipid deposition. In healthy humans, a four-week HFrD alters lipid homoeostasis, but does not affect insulin sensitivity or intramyocellular lipids (IMCL). The aim of this study was to investigate whether fructose may induce early molecular changes in skeletal muscle prior to the development of whole-body insulin resistance. METHODS: Muscle biopsies were taken from five healthy men who had participated in a previous four-week HFrD study, during which insulin sensitivity (hyperinsulinaemic euglycaemic clamp), and intrahepatocellular lipids and IMCL were assessed before and after HFrD. The mRNA concentrations of 16 genes involved in lipid and carbohydrate metabolism were quantified before and after HFrD by real-time quantitative PCR. RESULTS: HFrD significantly (P<0.05) increased stearoyl-CoA desaturase-1 (SCD-1) (+50%). Glucose transporter-4 (GLUT-4) decreased by 27% and acetyl-CoA carboxylase-2 decreased by 48%. A trend toward decreased peroxisomal proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) was observed (-26%, P=0.06). All other genes showed no significant changes. CONCLUSION: HFrD led to alterations of SCD-1, GLUT-4 and PGC-1alpha, which may be early markers of insulin resistance.

Molecular genetics of growth and development in populus. IV. Mapping QTLs with large effects on growth, form, and phenology traits in a forest tree.

We have mapped quantitative trait loci (QTLs) for commercially important traits (stem growth and form) and an adaptive trait (spring leaf flush) in a Populus F2 generation derived from a cross between interspecific F1 hybrids (P. trichocarpa x P. deltoides). Phenotypic data were collected over a 2-year period from a replicated clonal trial containing ramets of the parental, F1, and F2 trees. Contrary to the assumptions of simple polygenic models of quantitative trait inheritance, 1-5 QTLs of large effect are responsible for a large portion of the genetic variance in each of the traits measured. For example, 44.7% of the genetic variance in stem volume after 2 years of growth is controlled by just two QTLs. QTLs governing stem basal area were found clustered with QTLs for sylleptic branch leaf area, sharing similar chromosomal position and mode of action and suggesting a pleiotropic effect of QTLs ultimately responsible for stem diameter growth.

Penicillin levels following the administration of benzathine penicillin G in pregnancy.

OBJECTIVE: To investigate the distribution of penicillin in the maternal-placental-fetal unit at term gestation. METHODS: Twenty-five healthy gravidas at 38-39 weeks' gestation scheduled for elective repeat cesarean delivery under spinal anesthesia received benzathine penicillin G, 2.4 million units intramuscularly (IM) preoperatively. Ten women delivered 1 day after injection, five delivered 2-3 days after, and ten delivered 7 days after. We collected maternal serum and cerebrospinal fluid, amniotic fluid (AF), and cord serum at delivery. Penicillin levels were measured using a validated agar disc diffusion method (sensitivity 0.006 micrograms/mL) with Micrococcus lutea as the test organism. RESULTS: There was no significant difference in mean penicillin levels at day 1, day 2-3, or day 7 for maternal serum, maternal cerebrospinal fluid, cord serum, or AF. The mean (+/- standard error) penicillin concentration (range 0.005-0.59 micrograms/mL) in maternal serum declined from 0.14 +/- 0.04 micrograms/mL 1 day after injection to 0.08 +/- 0.06 micrograms/mL 7 days after injection. The proportion of patients with a penicillin concentration at or above 0.018 micrograms/mL in the maternal serum declined significantly from day 1 to day 7 (P = .03). Overall, nine of 25 women (36%) had serum penicillin levels that were less than 0.018 micrograms/mL. CONCLUSION: A wide range of penicillin levels were observed in gravidas at term in the maternal serum, cerebrospinal fluid, umbilical cord serum, and AF within 1 week after 2.4 million units of benzathine penicillin G IM. We speculate that altered pharmacokinetics may affect the efficacy of this drug for prevention of congenital syphilis in the near-term gestation.

Seroprevalence and risk factors for hepatitis C virus antibody in pregnant women.

OBJECTIVE: To better understand hepatitis C viropathies and seroprevalence by performing an epidemiologic analysis of pregnant women seropositive for antibody against hepatitis C. METHODS: We studied 1013 consecutive obstetric patients at Parkland Memorial Hospital who gave informed consent for detailed interviews and serotesting. Sera were analyzed for antibody to the hepatitis C virus using both C100-3 and RIBA-4 assays. Neonatal assessment was carried out in the immediate postpartum period. RESULTS: Hepatitis C antibody was detected in 2.28% (N = 23) of the 1005 women in whom analysis was completed. Factors associated with seropositivity included intravenous (IV) drug use, sexually transmitted diseases, hepatitis B infection, maternal age greater than 22.5 years, increased parity (eg, greater than 2.1), history of transfusion, and three or more different lifetime sexual partners or a sexual partner who used IV drugs. Maternal and neonatal outcome was not different between hepatitis C antibody-positive and -negative groups. CONCLUSIONS: Epidemiologic data are consistent with sexual and parenteral modes of transmission. Women with hepatitis C antibody did not have excessive perinatal complications compared with antibody-negative women. A model protocol and cost analysis for screening pregnant women for hepatitis C infection are presented. However, routine screening for hepatitis C is not advocated.

Evidence for a defective prophage on the chromosome of Methanobacterium wolfei.

Evidence shows the presence on the chromosome of Methanobacterium wolfei of a defective prophage which, by DNA-DNA hybridization, is closely related to the virulent archaeophage psi M1 of Methanobacterium thermoautotrophicum Marburg. Partial sequencing of a M. wolfei 16S rRNA gene and phylogenetic analysis indicated that this organism is more closely related to other representatives of the genus Methanobacterium than to M. thermoautotrophicum Marburg. The chromosomal region of M. wolfei encoding the putative prophage was found to be deleted for two non-contiguous segments of the phage psi M1 genome and thus encompassed only 80 to 90% of the psi M1 DNA. The prophage region was mapped to a 30 kb restriction fragment on the physical map of the M. wolfei chromosome. A randomly chosen DNA fragment was cloned from phage psi M1 DNA, as was its homologous counterpart from the chromosome of M. wolfei. The 126-bp region present in both clones exhibited 100% sequence identity.

Physical map of the Methanobacterium thermoautotrophicum Marburg chromosome.

A physical map of the Methanobacterium thermoautotrophicum Marburg chromosome was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by NotI, PmeI, and NheI. The order of the fragments was deduced from Southern blot hybridization of NotI fragment probes to various restriction digests and from partial digests. The derived map is circular, and the genome size was estimated to be 1,623 kb. Several cloned genes were hybridized to restriction fragments to locate their positions on the map. Genes coding for proteins involved in the methanogenic pathway were located on the same segment of the circular chromosome. In addition, the genomes of a variety of thermophilic Methanobacterium strains were treated with restriction enzymes and analyzed by pulsed-field gel electrophoresis. The sums of the fragment sizes varied from 1,600 to 1,728 kb among the strains, and widely different macrorestriction patterns were observed.

Natural history of chronic proteinuria complicating pregnancy.

OBJECTIVE: Although the significance of proteinuria is well-documented for pregnancy complicated by preeclampsia or diabetes, protein excretion of up to 300 mg per day is considered normal for uncomplicated pregnancy. Our purpose was to determine the significance of otherwise "asymptomatic" proteinuria identified during pregnancy. STUDY DESIGN: We reviewed the perinatal outcome of 65 pregnancies in 53 women with the following criteria: (1) proteinuria exceeding 500 mg per day, (2) no previously known renal disease, (3) no reversible renal dysfunction, and (4) no evidence for preeclampsia at discovery. RESULTS: Renal insufficiency coexisted in 62% of women, and 40% had chronic hypertension. Excluding 8 abortions, 53 (93%) of 57 pregnancies resulted in live infants; 45% of infants were delivered preterm and 23% had growth retardation. Of these 57 women, 62% demonstrated clinical evidence compatible with superimposed preeclampsia, and although the incidence of preeclampsia was increased with isolated proteinuria (29%), it was increased even more when there was associated chronic hypertension (incidence 100%) or renal insufficiency (incidence 58%). All 21 women who eventually underwent renal biopsy had histologic evidence of renal disease. To date, with only a limited follow-up of these 53 women, 11 (20%) have progressed to end-stage renal disease. CONCLUSION: "Asymptomatic" proteinuria is associated with a number of adverse pregnancy outcomes and serious long-term maternal morbidity.

Molecular genetics of growth and development in Populus. II. Segregation distortion due to genetic load.

Distortion of expected Mendelian segregation ratios, commonly observed in many plant taxa, has been detected in an experimental three-generation inbred pedigree of Populus founded by interspecific hybridization between P. trichocarpa and P. deltoides. An RFLP linkage map was constructed around a single locus showing severe skewing of segregation ratio against F2 trees carrying the P. trichocarpa allele in homozygous form. Several hypotheses for the mechanism of segregation distortion at this locus were tested, including directional chromosome loss, segregation of a pollen lethal allele, conflicts between genetic factors that isolate the parental species, and inbreeding depression as a result of genetic load. Breeding experiments to produce inbred and outcrossed progenies were combined with PCR-based detection of RFLPs to follow the fate of the deficient allele throughout embryo and seedling development. A recessive lethal allele, lth, inherited from the P. trichocarpa parent, was found to be tightly linked to the RFLP marker locus POP1054 and to cause embryo and seedling mortality. Heterozygotes (lth/+) appear to be phenotypically normal as embryos, seedlings, and young trees.

Quantitative genetics of growth and development in Populus. I. A three-generation comparison of tree architecture during the first 2 years of growth.

One approach to gain an insight into the genetics of tree architecture is to make use of morphologically divergent parents and study their segregating progeny in the F2 and backcross (B1) generations. This approach was chosen in the present study in which material of a three-generation pedigree growing side by side in a replicated plantation, was analyzed. The pedigree included Populus trichocarpa (T) and P. deltoides (D) parents, their F1 and F2 hybrids and their B1 hybrids to the D parent. The trees were grown in the environment of the T parent and measured for the first 2 years of growth. Nine quantitative traits were studied at the stem, branch and leaf levels of tree architecture, in which the original parents differed. Strong F1 hybrid vigor relative to the better parent (T) was expressed in growth and its components. Most quantitative traits in the F2 and B1 hybrids were intermediate between the T and D parents but displayed a wide range of variation due to segregation. The results from the analysis of variance indicated that all morphometric traits were significantly different among F2 and B1 clones, but the B1 hybrids were more sensitive to replicates than the F2. Broad-sense heritabilities (H (2)) based on clonal means ranged from moderately high to high (0.50-0.90) for the traits studied, with H (2) values varying over age. The H (2) estimates reflected greater environmental "noise" in the B1 than in the F2, presumably due to the greater proportion of maladaptive D alleles in those hybrids. In both families, sylleptic branch number and length, and leaf size on the terminal, showed strong genetic correlations with stem growth. The large divergence between the two original parents in the traits studied, combined with the high chromosome number in Populus (2n=38), makes this pedigree well suited for the estimation of the number of quantitative trait loci (QTLs) underlying quantitative variation by Wright's biometric method (1968). Variation in several traits was found to be under the control of surprisingly few major QTLs: 3-4 in 2nd-year height and diameter growth, a single QTL in stem diameter/height ratio.

The choice of genetic material for mechanistic studies of adaptation in forest trees.

There is considerable urgency to study the mechanisms by which forest trees track environmental change, given the prospect of possible rapid climatic changes. Environmental tracking is achieved through three basic processes: (1) expression of phenotypic plasticity at the level of the individual; and (2) evolutionary change and (3) migration, both expressed at the level of the population over generations. The current distribution and genetic architecture of a species reflect how these processes interacted in response to past climatic changes during and after the last glaciation. Part of that record is encoded in the DNA of the current generation of trees and, as a result of existing field tests, is accessible for study. These field tests include, in ascending order of genetic resolution, (1) provenance tests, (2) progeny tests, and (3) three-generation clonal tests; as well as (4) clone tests, with or without genetic structure. The suitability and limitations of these tests for mechanistic studies of environmental tracking are described, both as field installations and as sources of material for parallel in-depth studies. We conclude that they represent an important information resource, which deserves to be more effectively used by the scientific community.

[Accidental insertion of a temporary pacemaker electrode in the left ventricle: radiological and electrocardiographic diagnosis]. / Akzidentelle Einlage einer passageren Schrittmacherelektrode in den linken Ventrikel: radiologische und echokardiographische Diagnose.

We report a case of unintentional puncture of the subclavian artery and insertion of a temporary pacemaker electrode in the left ventricle. Therapeutic and diagnostic strategies of this rare complication of pacemaker therapy are discussed and recommendations for safe puncture of the subclavian vein are made.

Molecular genetics of growth and development in Populus. I. Triploidy in hybrid poplars.

While constructing a genetic linkage map of a hybrid poplar genome (Populus trichocarpa x P. deltoides), we identified several restriction fragment length polymorphismus (RFLPs) for which the parental trees are heterozygous. Although 8 of the 11 F1 hybrid offspring inherited, as expected, single RFLP alleles from each parent, 3 F1 trees in the mapping pedigree inherited both maternal alleles along with a single paternal allele at some loci. Aneuploidy or polyploidy in these 3 F1 trees due to partial or complete nondisj unction during female gametogenesis is the simplest explanation for this finding. Of the 3 f1 offspring with supernumerary RFLP alleles 2 have triploid nuclear DNA contents as measured by fluorescence flow cytometry; the 3rd F1 with supernumerary alleles has a sub-triploid nuclear DNA content and is probably aneuploid. Among the tri/aneuploid hybrids, leaf quantitative traits either are skewed toward those values characteristic of the P. trichocarpa female parent (adaxial stomate density, petiole length: blade length ratio; abaxial color) or show transgressive variation (epidermal cell size). Abaxial leaf color was used to screen a large population of P. trichocarpa x P. deltoides hybrids for further evidence of tri/aneuploidy. In each case where a "white" abaxial leaf surface was observed and the nuclear DNA content measured, the hybrid proved to be tri/aneuploid. All sexually mature female triploids examined were sterile, although the inflorescences completed their development in the absence of embryo formation. The (probably) aneuploid F1 hybrid is a fertile female. Of 15 female P. trichocarpa parents used in crosses to P. deltoides, 10 produced one or more tri/aneuploid hybrid offspring. In an intraspecific cross using a P. trichocarpa female that had produced triploid hybrids with five different P. deltoides males, no tri/aneuploid offpsring were found.

Molecular genetics of growth and development in Populus (Salicaceae). v. mapping quantitative trait loci affecting leaf variation.

We examined the genetic variation of leaf morphology and development in the 2-yr-old replicated plantation of an interspecific hybrid pedigree of Populus trichocarpa T. & G. and P. deltoides Marsh. via both molecular and quantitative genetic methods. Leaf traits chosen were those that show pronounced differences between the original parents, including leaf size, shape, orientation, color, structure, petiole size, and petiole cross section. Leaves were sampled from the current terminal, proleptic, and sylleptic branches. In the F2 generation, leaf traits were all significantly different among genotypes, but with significant effects due to genotype X crown-position interaction. Variation in leaf pigmentation, petiole length. And petiole length proportion appeared to be under the control of few quantitative trait loci (QTLs). More QTLs were associated with single leaf area, leaf shape, lamina angle, abaxial color, and petiole flatness, and in these traits the number of QTLs varied among crown positions. In general, the estimates of QTL numbers from Wright's biometric method were close to those derived from molecular markers. For those traits with few underlying QTLs, a single marker interval could explain from 30 to 60% of the observed phenotypic variance. For multigenic traits, certain markers contributed more substantially to the observed variation than others. Genetic cluster analysis showed developmentally related traits to be more strongly associated with each other than with unrelated traits. This finding was also supported by the QTL mapping. For example, the same chromosomal segment of linkage group L seemed to account for 20% of the phenotypic variation of all dimension-related traits, leaf size, petiole length. and midrib angle. In both traits. the P. deltoides alleles had positive effects and were dominant to the P. trichocarpa alleles. Similar relationships were also found for lamina angle. abaxial greenness, and petiole.

Influence of soil water on the physiological and morphological components of plant water balance in Populus trichocarpa, Populus deltoides and their F(1) hybrids.

Patterns of leaf growth, transpiration and whole-plant water balance in Populus trichocarpa, P. deltoides and their F(1) hybrids were studied during a soil drying cycle. Plant responses were analyzed during three distinct stages of dehydration. In stage I, the transpiration rate of drought-stressed plants remained constant and equal to that of well-watered plants even though soil water content declined by more than 40%. Stage II began as soil and plant water deficits induced stomatal closure. When soil water was expressed as a fraction of transpirable soil water, the transition from stage I to stage II occurred at soil water fractions of 0.35, 0.45 and 0.60 for P. trichocarpa, P. deltoides and their F(1) hybrids, respectively. Reductions in leaf growth coincided with the shift from stage I to stage II. As soil water declined further, decreases in relative transpiration and whole-plant leaf area were significantly greater in parental species than in F(1) hybrids. Inherent feedbacks controlling stomatal water loss and the maintenance and growth of leaf tissue appeared to differ between F(1) and parental genotypes in a pattern characteristic of an overdominant mode of inheritance.Stage III began once the ability of stomata to compensate for water loss had been exhausted. Substantial differences were found in plant survival during stage III, with F(1) hybrids surviving longer than parental species. Survival was more strongly correlated with the hydraulic conductivity of xylem tissues than with the dehydration tolerance of leaf tissues. Collectively, these responses suggest that F(1) hybrids were more drought resistant than either parental species and highlight the importance of whole-plant studies of functional relationships between plant growth, water balance and hydraulic conductivity.

Interspecific crossability studies in poplars.

Pollen-stigma interactions were studied with scanning electron microscopy in intraspecific and intersectional crosses of Populus tremuloides (Sect. LEUCE); P. deltoides, P. nigra (Sect. AIGEIROS); and P. trichocarpa (Sect. TACAMAHACA). Intraspecific variation in hydration, size of hydrated pollen grains, and tube morphology is described. Exine sculpture in P. tremuloides was densely spinulate or scabrate, in P. trichocarpa densely reticulate, in the other species somewhat intermediate. In six of seven intersectional cross combinations pollen-tube behavior on the stigmatic surface at 5-22 hours after pollination was the same as in conspecific matings; in P. tremuloides x P. trichocarpa crosses, the percentage of twisted and bulging tubes was significantly higher. Irradiated mentor pollen did not affect pollentube behavior of adjacent incompatible pollen, but increased hybrid seedling production in interspecific crosses. By contrast, hexane treatment of pollen or stigma, and the spraying of catkins with gibberellic acid or naphtalene acetic acid were ineffective. The results are discussed in relation to several mechanisms presumed to be involved in restricting gene exchange among poplar species; and in relation to Hogenboom's (1973) incongruity concept.

Seasonal and spatial community dynamics in the meromictic Lake Cadagno.

The seasonal and spatial variations in the community structure of bacterioplankton in the meromictic alpine Lake Cadagno were examined by temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rDNA fragments. Two different amplifications were performed, one specific for the domain Bacteria (Escherichia coli positions 8-536) and another specific for the family Chromatiaceae (E. coli positions 8-1005). The latter was followed by semi-nested reamplification with the bacterial primer set, allowing comparison of the two PCR approaches by TTGE. The TTGE patterns of samples from the chemocline and the anoxic monimolimnion were essentially identical, whereas the oxic mixolimnion displayed distinctively different banding patterns. For samples from the chemocline and the monimolimnion, dominant bands in the Bacteria-specific TTGE profiles comigrated with bands obtained by the semi-nested PCR approach specific for Chromatiaceae. This observation suggested that Chromatiaceae are in high abundance in the anoxic water layer. All dominant bands were excised and sequenced. Changes in the community structure, as indicated by changes in the TTGE profiles, were observed in samples taken at different times of the year. In the chemocline, Chomatium okenii was dominant in the summer months, whereas Amoebobacter purpureus populations dominated in autumn and winter. This change was confirmed by fluorescent in situ hybridization.

Molecular analysis of Methanobacterium phage psiM2.

The methanogenic archaeon Methanobacterium thermoautotrophicum Marburg is infected by the double-stranded DNA phage psiM2. The complete phage genome sequence of 26 111 bp was established. Thirty-one open reading frames (orfs), all of them organized in the same direction of transcription, were identified. On the basis of comparison of the deduced amino acid sequences to known proteins and by searching for conserved motifs, putative functions were assigned to the products of six orfs. These included three proteins involved in packaging DNA into the capsid, two putative phage structural proteins and a protein related to the Int family of site-specific recombinases. Analysis of the N-terminal amino acid sequences of three phage-encoded proteins led to the identification of two genes encoding structural proteins and of peiP, the structural gene of pseudomurein endoisopeptidase. This enzyme is involved in the lysis of host cells, and it appears to belong to a novel enzyme family. peiP was overexpressed in Escherichia coli, and its product was shown to catalyse the in vitro lysis of M. thermoautotrophicum cells. Comparison of the phage psiM2 DNA sequence with parts of the sequence of the wild-type phage psiM1 suggests that psiM2 is a deletion derivative, which formed by homologous recombination between two copies of a direct repeat.

Aerobic mineralization of 2,6-dichlorophenol by Ralstonia sp. strain RK1.

A new aerobic bacterium was isolated from the sediment of a freshwater pond close to a contaminated site at Amponville (France). It was enriched in a fixed-bed reactor fed with 2,6-dichlorophenol (2,6-DCP)as the sole carbon and energy source at pH 7.5 and room temperature. The degradation of 2,6-DCP followed Monod kinetics at low initial concentrations. At concentrations above 300 microM (50 mg.liter-1), 2,6-DCP increasingly inhibited its own degradation. The base sequence of the 16S ribosomal DNA allowed us to assign the bacterium to the genus Ralstonia (formerly Alcaligenes). The substrate spectrum of the bacterium includes toluene, benzene, chlorobenzene, phenol, and all four ortho- and para-substituted mono- and dichlorophenol isomers. Substituents other than chlorine prevented degradation. The capacity to degrade 2,6-DCP was examined in two fixed-bed reactors. The microbial population grew on and completely mineralized 2,6-DCP at 2,6-DCP concentrations up to 740 microM in continuous reactor culture supplied with H2O2 as an oxygen source. Lack of peroxide completely stopped further degradation of 2,6-DCP. Lowering the acid-neutralizing capacity of the medium to 1/10th the original capacity led to a decrease in the pH of the effluent from 7 to 6 and to a significant reduction in the degradation activity. A second fixed-bed reactor successfully removed low chlorophenol concentrations (20 to 26 microM) with hydraulic residence times of 8 to 30 min.

Tryptophan gene cluster of Methanobacterium thermoautotrophicum Marburg: molecular cloning and nucleotide sequence of a putative trpEGCFBAD operon.

A recombinant cosmid carrying the Methanobacterium thermoautotrophicum Marburg trp genes was selected by complementation of Escherichia coli trp mutations. A 7.3-kb fragment of the cloned archaeal DNA was sequenced. It contained the seven trp genes, arranged adjacent to each other in the order trpEGCFBAD. No gene fusions were observed. The trp genes were organized in an operonlike structure, with four short (5- to 56-bp) intergenic regions and two overlapping genes. There was no indication for an open reading frame encoding a leader peptide in the upstream region of trpE. The gene order observed in the M. thermoautotrophicum trp operon was different from all known arrangements of the trp genes in archaea, bacteria, and eucarya. The encoded sequences of the Methanobacterium Trp proteins were similar in size to their bacterial and eucaryal counterparts, and all of them contained the segments of highly similar or invariant amino acid residues recognized in the Trp enzymes from bacteria and eucarya. The TrpE, TrpG, TrpC, TrpA, and TrpD proteins were 30 to 50% identical to those from representatives of other species. Significantly less sequence conservation (18 to 30%) was observed for TrpF, and TrpB exhibited a high degree of identity (50 to 62%) to the sequences of representatives of the three domains. With the exception of TrpB, the beta subunit of tryptophan synthase, tryptophan was absent from all Trp polypeptides.