Time to reconsider the role of ribavirin in Lassa fever

Ribavirin is the only available Lassa fever treatment. The rationale for using ribavirin is based on one clinical study conducted in the early 1980s. However, reanalysis of previous unpublished data reveals that ribavirin may actually be harmful in some Lassa fever patients. An urgent reevaluation of ribavirin is therefore needed.

Fifty years after its discovery, Lassa fever remains uncontrolled, and mortality remains unacceptably high. Since 2015, Nigeria has been experiencing increasingly large outbreaks of Lassa fever, with new peaks reached in 2016, 2017, and 2018. In 1987, McCormick and colleagues reported a case fatality rate (CFR) of 16.5% among 441 patients hospitalized in Sierra Leone [1]. In Nigeria in 2019, 124 deaths were recorded among 554 laboratory-confirmed cases for a CFR of 22% [2].Ribavirin is the only available Lassa fever–specific treatment and has been used routinely for over 25 years. However, intravenous ribavirin is not licensed for Lassa fever. Its mechanism of action is unclear, it is expensive and hard to source, and it has well-known toxicities [3]. Therefore, the evidence for using ribavirin in Lassa fever deserves careful scrutiny. The emergence of potential new therapeutics for Lassa fever, such as favipiravir and monoclonal antibodies, adds further weight to the case for reconsidering the role of ribavirin since the evaluation of new drugs in clinical trials requires a comparison against existing treatments with a known efficacy and safety profile [4,5].The rationale for using ribavirin in Lassa fever is primarily based on one clinical study conducted in Sierra Leone in the late 1970s and early 1980s. McCormick and colleagues [6] reported that in Lassa fever patients with a serum aspartate aminotransferase (AST) level of ≥150 IU/L, the use of intravenous ribavirin within the first 6 days of illness reduced the fatality rate from 61% (11/18) with no ribavirin to 5% (1/20) (p = 0.002). These authors concluded that ribavirin is effective in the treatment of Lassa fever. However, there are long-standing concerns about the methods used in this study. Although randomization was used to assign patients to treatment groups, the comparisons presented were not according to original randomized groups, and we have reconstructed their derivation (Fig 1). Serious limitations to the comparisons presented include the use of historic controls, inclusion of pregnant women in the control group but their exclusion from the ribavirin group (case fatality is around 2-fold higher in pregnant women than nonpregnant patients), and post hoc merging of treatment groups. Despite this and the fact that the results only supported the use of ribavirin in nonpregnant adult patients with AST ≥150 IU/L, this study is the basis upon which ribavirin is now used in all patients with Lassa fever, including children, pregnant women, and people with normal liver function.Open in a separate windowFig 1Reconstruction of the McCormick et al. data.AST, aspartate aminotransferase; PW, pregnant women. † Discrepancy within McCormick et al, with 39 patients reported treated with oral ribavirin but only 38 (14+24) outcomes reported. ‡ Discrepancy within McCormick et al, with table 1 reporting 12/63 but text reporting 13/62.It has been well known among Lassa specialists that the McCormick study reports a subset of a much larger dataset assembled by the Lassa treatment unit in Sierra Leone and that a report on the full dataset was commissioned by the United States Army Medical Research and Development Command. One of us (PH) therefore submitted a freedom of information (FOI) request to access this report. The full report and an accompanying memo are available, and we encourage readers to access and read the materials [7,8]. The memo states that some of the original trial records were unavailable, and the data should be “interpreted with extreme caution.” Nonetheless, the report presents data from 1977 through to 1991 on 807 Lassa fever patients with a known outcome that were assigned to different ribavirin treatment regimens. These newly available data raise important questions about the safety and efficacy of ribavirin for the treatment of Lassa fever.The original data were lost during the civil war in Sierra Leone, but the report contains tables showing the distribution of characteristics of the whole population according to treatment group, an appendix showing individual data for the 405 patients who died, and results of a logistic regression analysis comparing the effect of ribavirin with no treatment for some of the ribavirin regimens, after adjusting for patient characteristics. Based on these data, we derived aggregated datasets containing the number of deaths according to treatment groups and individual characteristics. We combined groups I (“No treatment given”) and X (“Drugs were not available”) as no treatment and all groups in which ribavirin was administered (II, III, and V to IX) as ribavirin. Exhibit III-8 in the FOI report presented case fatality by treatment group and AST, from which we derived crude odds ratios (ORs) comparing ribavirin with no treatment. The logistic regression reported in Exhibit III-9 was restricted to “those treatment groups that yielded the lowest case fatality rates with respect to untreated patients in the high severity patient illness category” (groups II, III, V, and VII). It was adjusted for age, gender, time to admission, time to treatment, length of stay, and log(AST). We also reconstructed analyses by digitizing the data on individuals who died in Appendix D, calculating the number of deaths according to treatment group and AST, and subtracting these numbers from the totals presented in Exhibit III-2. These allowed us to estimate overall mortality ORs before and after adjusting for ribavirin, although the numbers did not entirely match, and so the number of deaths was reduced in some small groups.Estimates of the effect of oral and intravenous ribavirin from the McCormick study and of all ribavirin from the full report are shown in Fig 2. Based on the crude ORs derived from Exhibit III-8, ribavirin reduced mortality only in patients with serum AST ≥150 IU/L, with less benefit (OR 0.48 [95% CI 0.30 to 0.78]) than reported by McCormick and colleagues. However, ribavirin appeared to increase mortality in patients with serum AST <150 IU/L (2.90 [1.42 to 5.95]). In fact, in our analysis, the only stratum in which ribavirin appeared protective (0.38 [0.21 to 0.70]) was serum AST >300 IU/L (Table H in S1 Text). The logistic regression reported in the FOI report suggested a modest reduction in mortality, but the reasons for the choice of treatment groups compared were unclear. In the reconstructed analyses, ribavirin was associated with overall increased mortality (2.12 [1.67, 2.68]), although this was attenuated after adjustment for AST (1.48 [1.05, 2.08]).Open in a separate windowFig 2Forest plot of the OR of death in treatment and risk subgroups.AST, aspartate aminotransferase; FOI, freedom of information; OR, odds ratio.In our view, there is a compelling case to reevaluate the role of ribavirin in the care of patients with Lassa fever. The data suggest that ribavirin treatment may harm Lassa fever patients with AST <150 IU/L. The limitations revealed by the US Army report, such as large amounts of missing data, unclear treatment allocation practices, imbalances in treatment groups, and errors in coding serology results, cast further doubt on the conclusions of the McCormick study. This aligns with 2 recent systematic reviews by Eberhardt and colleagues and Cheng and colleagues, which concluded that the efficacy of ribavirin in Lassa fever was uncertain because of critical risk of bias in existing studies [9,10].Challenging a quarter of century of clinical practice is difficult. The first step is to acknowledge inadequacies in our knowledge and to ensure that treatment recommendations for Lassa fever better reflect the (weak) strength of evidence for ribavirin in different patient populations. Vigorous efforts should be made to engage clinicians and patients in designing a placebo-controlled trial to assess the safety and efficacy of ribavirin treatment in Lassa fever patients, particularly in those with milder disease (as may be indicated by an admission AST <150 IU/L) in whom the available evidence is compatible with ribavirin causing more harm than good.In conclusion, Lassa fever patients are receiving a drug that may lack efficacy or cause harm. It is incumbent on us to ensure that the next 25 years of Lassa fever treatment are built on more solid foundations.     

The Ras‐ERK pathway: Understanding site‐specific signaling provides hope of new anti‐tumor therapies

Recent discoveries have suggested the concept that intracellular signals are the sum of multiple, site‐specified subsignals, rather than single, homogeneous entities. In the context of cancer, searching for compounds that selectively block subsignals essential for tumor progression, but not those regulating “house‐keeping” functions, could help in producing drugs with reduced side effects compared to compounds that block signaling completely. The Ras‐ERK pathway has become a paradigm of how space can differentially shape signaling. Today, we know that Ras proteins are found in different plasma membrane microdomains and endomembranes. At these localizations, Ras is subject to site‐specific regulatory mechanisms, distinctively engaging effector pathways and switching‐on diverse genetic programs to generate different biological responses. The Ras effector pathway leading to ERKs activation is also under strict, space‐related regulatory processes. These findings may open a gate for aiming at the Ras‐ERK pathway in a spatially restricted fashion, in our quest for new anti‐tumor therapies.     

Interactions between quercetin and Warfarin for albumin binding: A new eye on food/drug interference

The interaction between quercetin, a popular antioxidant flavonoid, and human serum albumin (HSA) is investigated and characterized by means of induced circular dichroism and saturation transfer difference NMR. These techiques demonstrate the reversible binding of quercetin to the carrier protein, which is responsible for its dissolution in aqueous medium. Competition experiments with two classical probes for HSA binding sites, namely Ibuprofen and Warfarin (a common anticoagulant coumarin), demonstrate that quercetin has a primary binding site located in the subdomain IIA, where coumarins are hosted. The affinity for this site is large and we found that quercetin may effectively displace warfarin from HSA. This may have relevant consequences in rationalizing the interferences of common dietary compounds and food supplements to anticoagulant treatments. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Biodiversity of Phototrophic Biofilms Dwelling on Monumental Fountains

Among the stone monumental assets, artistic fountains are particularly affected by microbial colonization due to constant contact with water, giving rise to biodegradation processes related with physical–chemical and aesthetical alterations. In this paper, we make an overview of reported biodiversity of the phototrophic patina developed in various fountains of Italy and Spain. The microbial composition of four fountains (two from Florence, Italy and two from Granada, Spain) was investigated using traditional and/or molecular techniques. The results indicated many common similarities with regard the phototrophic biodiversity for all the investigated fountains. Automated ribosomal RNA intergenic spacer analysis (ARISA), a molecular fingerprint tool, was used to examine the eubacterial and cyanobacterial community for two of the investigated fountains. The principal component analysis of ARISA profiles strengthens the results obtained by traditional methods and revealed separate clusters, as a consequence of the differences of micro-environmental conditions for each fountain.     

Pim-1 regulates cardiomyocyte survival downstream of Akt

The serine-threonine kinases Pim-1 and Akt regulate cellular proliferation and survival. Although Akt is known to be a crucial signaling protein in the myocardium, the role of Pim-1 has been overlooked. Pim-1 expression in the myocardium of mice decreased during postnatal development, re-emerged after acute pathological injury in mice and was increased in failing hearts of both mice and humans. Cardioprotective stimuli associated with Akt activation induced Pim-1 expression, but compensatory increases in Akt abundance and phosphorylation after pathological injury by infarction or pressure overload did not protect the myocardium in Pim-1-deficient mice. Transgenic expression of Pim-1 in the myocardium protected mice from infarction injury, and Pim-1 expression inhibited cardiomyocyte apoptosis with concomitant increases in Bcl-2 and Bcl-X(L) protein levels, as well as in Bad phosphorylation levels. Relative to nontransgenic controls, calcium dynamics were significantly enhanced in Pim-1-overexpressing transgenic hearts, associated with increased expression of SERCA2a, and were depressed in Pim-1-deficient hearts. Collectively, these data suggest that Pim-1 is a crucial facet of cardioprotection downstream of Akt.     

Interrogating cAMP-dependent Kinase Signaling in Jurkat T Cells via a Protein Kinase A Targeted Immune-precipitation Phosphoproteomics Approach

In the past decade, mass-spectrometry-based methods have emerged for the quantitative profiling of dynamic changes in protein phosphorylation, allowing the behavior of thousands of phosphorylation sites to be monitored in a single experiment. However, when one is interested in specific signaling pathways, such shotgun methodologies are not ideal because they lack selectivity and are not cost and time efficient with respect to instrument and data analysis time.Here we evaluate and explore a peptide-centric antibody generated to selectively enrich peptides containing the cAMP-dependent protein kinase (PKA) consensus motif. This targeted phosphoproteomic strategy is used to profile temporal quantitative changes of potential PKA substrates in Jurkat T lymphocytes upon prostaglandin E₂ (PGE₂) stimulation, which increases intracellular cAMP, activating PKA. Our method combines ultra-high-specificity motif-based immunoaffinity purification with cost-efficient stable isotope dimethyl labeling. We identified 655 phosphopeptides, of which 642 (i.e. 98%) contained the consensus motif [R/K][R/K/X]X[pS/pT]. When our data were compared with a large-scale Jurkat T-lymphocyte phosphoproteomics dataset containing more than 10,500 phosphosites, a minimal overlap of 0.2% was observed. This stresses the need for such targeted analyses when the interest is in a particular kinase.Our data provide a resource of likely substrates of PKA, and potentially some substrates of closely related kinases. Network analysis revealed that about half of the observed substrates have been implicated in cAMP-induced signaling. Still, the other half of the here-identified substrates have been less well characterized, representing a valuable resource for future research.The identification and quantification of protein phosphorylation under system perturbations is an integral part of systems biology (1, 2). The combination of phosphopeptide enrichment (3–6), stable isotope labeling, and high-resolution mass spectrometry (MS) methods (7–9) has become the method of choice for the identification of novel phosphorylation sites and for the quantitation of temporal dynamics within signaling networks (10, 11), allowing the behavior of thousands of phosphorylation sites to be studied in a single experiment (10, 12, 13). Nowadays, one of the most commonly adopted high-throughput phosphoproteomics strategies utilizes two consecutive separation steps: (i) an initial fractionation to reduce the sample complexity, and (ii) a phosphopeptide-specific affinity purification. Such techniques include strong cation exchange fractionation under acidic conditions (3), followed by a chelation-based method with the use of metal ions (i.e. immobilized metal ion affinity chromatography (4), metal oxide affinity chromatography (10, 14), or Ti⁴⁺ immobilized metal ion affinity chromatography (6)). Alternatives to strong cation exchange for the first sample fractionation step have also been reported, including the use of electrostatic repulsion liquid chromatography (15, 16), which is well suited for the identification of multiply phosphorylated peptides, or hydrophilic interaction chromatography (17).Although the number of detected phosphorylated peptides is nowadays impressive, these kinds of methodologies are still inclined to identify/quantify the more abundant phosphoproteins present in a sample. For example, phosphotyrosine peptides are underrepresented because of their relatively lower abundance.In order to analyze key signaling events that may occur on less abundant phosphoproteins, more targeted approaches, focused on a specific pathway or a specific post-translational modification, are thus still essential. Studies examining post-translational modifications are often based on immunoaffinity purification at the protein or peptide level using dedicated antibodies. Recent examples include the selective enrichment of acetylated lysines (18) and phosphorylated tyrosines (19, 20). More recently, the first specific methods targeting serine/threonine phosphorylation motifs using immune-affinity assays have emerged (21, 22). The advantages of targeted approaches are their potentially higher sensitivity and more specific throughput with, as a consequence, relatively faster and easier data interpretation, which make them attractive for many systems biology applications.Immunoaffinity enrichment can be applied at both the protein and the peptide level, and both have been explored to study protein tyrosine phosphorylation (23). The first one results mainly in information on total protein phosphorylation levels. The detection of the actual phosphoresidue might be hampered by the high content of unmodified peptides derived from the immune-purified phosphoprotein and its binding partners. Immunoprecipitation at the peptide level (20, 24, 25), in contrast, leads to improved phosphosite characterization, with the identification of hundreds of sites, albeit with the loss (generally) of information regarding total protein expression.To profile the dynamic regulation of phosphorylation events via mass spectrometry, stable isotope labeling is often implemented, either with the use of amino acids in cell culture (10) or via chemical peptide labeling of the proteolytic digests (26, 27). To identify low-abundant signaling events, phosphoprotein/phosphopeptide immunoprecipitation is typically performed on several milligrams of material because of the substoichiometric abundance of post-translational modifications. This may hamper the use of expensive isotope-labeling reagents such as iTRAQ or tandem mass tag reagents, given the large amount of chemicals needed. Boersema et al. (28) introduced an alternative sensitive and accurate triplex labeling approach using inexpensive reagents (i.e. formaldehyde) that is much less limited in terms of the sample type or amount. We combined this latter stable-isotope dimethyl labeling approach (27–29) with highly specific antibodies raised against a set of cAMP-dependent protein kinase (PKA) phosphorylated substrates as based on the current literature (11, 30–34). It is generally accepted that PKA phosphorylates sites with the reasonably stringent consensus motif [R/K][R/K/X]X[pS/pT]. It should be noted that this consensus motif resembles somewhat the motifs of other AGC kinases (e.g. Akt, PKG, PKC).The basicity of the PKA motifs may hamper their analysis via MS-based proteomics, especially when trypsin is used as a protease, as the peptides may become too small to be sequenced. The use of trypsin is also unfavorable in the approach presented here when attempting to immunoprecipitate peptides bearing the PKA motif. Therefore, we decided to use Lys-C in order to keep the (dominant (RRX[pS/pT])) phosphorylated motif intact. To enhance identification, we applied decision-tree MS/MS technology (9), which makes use of HCD and ETD for more efficient fragmentation, higher mass accuracy in tandem MS mode, and less background noise (35).We applied this method to screen the response of Jurkat T cells to prostaglandin E₂ (PGE₂) treatment. PGE₂ is a potent inflammatory mediator that plays an important role in several immune-regulatory actions (36). It is produced by many different cell types, including tumor cells, where carcinogenesis is associated with chronic inflammatory responses (37). PGE₂ signaling in T cells is initiated by its binding to the G protein–coupled receptors EP1, -2, -3, and -4. Signaling pathways that are initiated by PGE₂ are for the most part under control of the second messenger cyclic adenosine monophosphate (cAMP),¹ which is generated from ATP by adenylyl cyclase when PGE₂ binds to EP2 or EP4 receptors. One of the primary targets of cAMP is PKA—cAMP binding releases the catalytic subunit activating the kinase. In the current study, we efficiently enriched close to 650 phosphopeptides containing the [R/K][R/K/X]X[pS/pT] consensus motif. Almost all these sites were absent in a recently reported comprehensive phosphoproteomics dataset of Jurkat T cells (12), compiled using shotgun strong cation exchange–immobilized metal ion affinity chromatography analysis and containing ∼10,500 phosphorylation sites, illustrative of the complementarity and selectivity of our approach. The qualitative and quantitative data presented here provide a wide-ranging and credible resource of likely PKA substrates. Network analysis confirmed several established cAMP-dependent signaling nodes in our dataset, although most identified potential PKA substrates are “novel” (i.e. not previously reported and/or linked to PKA). Therefore, the dataset presented here can be considered as a comprehensive and reliable resource for future research into cAMP-related signaling.     

Prostaglandins mediate tonic contraction of the guinea pig and human gallbladder

The gallbladder (GB) maintains tonic contraction modulated by neurohormonal inputs but generated by myogenic mechanisms. The aim of these studies was to examine the role of prostaglandins in the genesis of GB myogenic tension. Muscle strips and cells were treated with prostaglandin agonists, antagonists, cyclooxygenase (COX) inhibitors, and small interference RNA (siRNA). The results show that PGE2, thromboxane A2 (TxA2), and PGF(2alpha) cause a dose-dependent contraction of muscle strips and cells. However, only TxA2 and PGE2 (E prostanoid 1 receptor type) antagonists induced a dose-dependent decrease in tonic tension. A COX-1 inhibitor decreased partially the tonic contraction and TxB2 (TxA2 stable metabolite) levels; a COX-2 inhibitor lowered the tonic contraction partially and reduced PGE2 levels. Both inhibitors and the nonselective COX inhibitor indomethacin abolished the tonic contraction. Transfection of human GB muscle strips with COX-1 siRNA partially lowered the tonic contraction and reduced COX-1 protein expression and TxB2 levels; COX-2 siRNA also partially reduced the tonic contraction, the protein expression of COX-2, and PGE2. Stretching muscle strips by 1, 2, 3, and 4 g increased the active tension, TxB2, and PGE2 levels; a COX-1 inhibitor prevented the increase in tension and TxB2; and a COX-2 inhibitor inhibited the expected rise in tonic contraction and PGE2. Indomethacin blocked the rise in tension and TxB2 and PGE2 levels. We conclude that PGE2 generated by COX-2 and TxA2 generated by COX-1 contributes to the maintenance of GB tonic contraction and that variations in tonic contraction are associated with concomitant changes in PGE2 and TxA2 levels.     

Glutamate oxidative injury to RGC-5 cells in culture is necrostatin sensitive and blunted by a hydrogen sulfide (H2S)-releasing derivative of aspirin (ACS14)

Oxidative stress to RGC-5 cells in culture was delivered by exposure to a combination of glutamate (Glu) and buthionine-S,R-sulfoximine (BSO). The effect of the insult on cell survival was quantified by the resazurin-reduction and a dead/live assays. Moreover, breakdown of DNA, the localisation of phosphatidylserine and reactive radical species (ROS) and its quantification were determined. In addition, various proteins and mRNAs were studied using Western blot, real time PCR and immunocytochemistry. ACS14, its sulfurated moiety ACS1 and aspirin were tested for their ability to blunt the negative effects of Glu/BSO on RGC-5 cells. In addition assays were carried out to see whether any of these substances influenced glutathione (GSH). Glu/BSO dose-dependently kills RGC-5 cells by a mechanism that involves an elevation of ROS accompanied by a breakdown of DNA, expression of phosphatidylserine and the activation of p38 MAPK. The process is unaffected by the pan caspase inhibitor z-VAD-fmk, does not involve the activation of apoptosis inducing factor (AIF) but is sensitive to active necrostatin-1. In cell viability studies (resazurin-reduction assay), ACS1 and ACS14 equally counteracted the negative effects of 5mM Glu/BSO to RGC-5 cells but aspirin was only effective with a milder oxidative stress (1 mM Glu/BSO). In all other assays ACS14 was very much more effective than aspirin at counteracting the influence of 5mM Glu/BSO. Moreover, ACS14 and ACS1 directly stimulated GSH while aspirin was ineffective. In addition the neuroprotecive effect of ACS14 was specifically blunted by the non-specific potassium channel blocker glibenclamide. Also the up-regulation of Bcl-2, HO-1 and XIAP induced by 5mM Glu/BSO were all attenuated to a greater extent by ACS14 (20 μM) than aspirin (20 μM). These data show that ACS14 is a very effective neuroprotectant when compared with aspirin. ACS14 maintains its aspirin characteristics and has the ability to release H(2)S. The combined multiple actions of aspirin and H(2)S in the form of ACS14 is worthy to consider for possible use in the treatment of glaucoma.     

Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila,Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria

Ribosomal subunits of Caldariella acidophila (max.growth temp., 90°C) have been compared to subunits of Bacillus acidocaldarius (max. growth temp., 70°C) and Escherichia coli (max. growth temp., 47°C) with respect to (a) bihelical content of rRNA; (b) G·C content of bihelical domains and (c) tightness of rRNA-protein interactions. The principal results are as follows. 1. Subunits of C. acidophila ribosomes (T_m = 90–93°C) exhibit considerable thermal tolerance over their B. acidocaldarius (T_m = 77°C) and E. coli counterparts (T_m = 72°C). 2. Based on the ‘melting’ hyperchromicities of the intact ribosomal subunits a 51–55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67–70%, appears to be involved in hydrogen bonding in the naked rRNA species. 3. The G·C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C. acidophila rRNA are estimated to contain 63–64% G·C, compared to 58.5% G·C for B. acidocaldarius and 55% G·C for E. coli. 4. The increment in ribosome T_m values with increasing thermophily is greater than the increase in T_m for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s). 5. The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C. acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release rRNA from Celite-bound E. coli ribosomes. 6. Compared to E. coli, the C. acidophila 50 and 30 S ribosomal subunits are considerably less susceptible to treatment designed to promote ribosome unfolding through depletion of magnesium ions.     

Gibberellins in suspensor,embryo and integument from very young seeds of Phaseolus coccineus L.

Endogenous gibberellins (GAs) were extracted from suspensor, embryo and integument of very young seeds of Phaseolus coccineus L. and detected by combined gas chromatography-mass spectrometry (GC-MS). Results show the presence of one C₂₀-GA, GA₄₄ and five C₁₉-GAs in the suspensor: GA₁, GA₄, GA₅, GA₆ and GA₈, and four C₁₉-GAs in the integument: GA₁, GA₅, GA₆ and GA₈. Only traces of GA₁ and GA₅ were identified in the embryo. A compound structurally related to GAs was identified as tetrahydroxy-Kauranoic acid in suspensor, integument and, only in trace amounts, in the embryo.