Brettanomyces bruxellensis SSU1 Haplotypes Confer Different Levels of Sulfite Tolerance When Expressed in a Saccharomyces cerevisiae SSU1 Null Mutant.

The addition of SO2 is practiced in the wine industry to mitigate the risk of microbial spoilage and to extend wine shelf-life. Generally, this strategy does not interfere with primary alcoholic fermentation, as wine strains of Saccharomyces cerevisiae exhibit significant SO2 tolerance, largely driven by the efflux pump Ssu1p. One of the key yeast species responsible for wine spoilage is Brettanomyces bruxellensis, which also exhibits strain-dependent SO2 tolerance, although this occurs via unknown mechanisms. To evaluate the factors responsible for the differential sulfite tolerance observed in B. bruxellensis strains, we employed a multifaceted approach to examine both expression and allelic differences in the BbSSU1 gene. Transcriptomic analysis following exposure to SO2 highlighted different inducible responses in two B. bruxellensis strains. It also revealed disproportionate transcription of one putative BbSSU1 haplotype in both genetic backgrounds. Here, we confirm the functionality of BbSSU1 by complementation of a null mutant in a S. cerevisiae wine strain. The expression of four distinct BbSSU1 haplotypes in the S. cerevisiae ΔSSU1 mutant revealed up to a 3-fold difference in conferred SO2 tolerance. Substitution of key amino acids distinguishing the encoded proteins was performed to evaluate their relative contribution to SO2 tolerance. Protein modeling of two haplotypes which differed in two amino acid residues suggested that these substitutions affect the binding of Ssu1p ligands near the channel opening. Taken together, preferential transcription of a BbSSU1 allele that encodes a more efficient Ssu1p transporter may represent one mechanism that contributes to differences in sulfite tolerances between B. bruxellensis strains.IMPORTANCEBrettanomyces bruxellensis is one of the most important wine spoilage microorganisms, with the use of sulfite being the major method to control spoilage. However, this species displays a wide intraspecies distribution in sulfite tolerance, with some strains capable of tolerating high concentrations of SO2, with relatively high concentrations of this antimicrobial needed for their control. Although SO2 tolerance has been studied in several organisms and particularly in S. cerevisiae, little is known about the mechanisms that confer SO2 tolerance in B. bruxellensis Here, we confirmed the functionality of the sulfite efflux pump encoded by BbSSU1 and determined the efficiencies of four different BbSSU1 haplotypes. Gene expression analysis showed greater expression of the haplotype conferring greater SO2 tolerance. Our results suggest that a combination of BbSSU1 haplotype efficiency, copy number, and haplotype expression levels likely contributes to the diverse SO2 tolerances observed for different B. bruxellensis strains.

Oncostatin M promotes cancer cell plasticity through cooperative STAT3-SMAD3 signaling.

Increasing evidence supports the idea that cancer cell plasticity promotes metastasis and tumor recurrence, resulting in patient mortality. While it is clear that the tumor microenvironment (TME) contributes to cancer cell plasticity, the specific TME factors most actively controlling plasticity remain largely unknown. Here, we performed a screen to identify TME cytokines and growth factors that promote epithelial-mesenchymal plasticity, and acquisition of cancer stem cell (CSC) properties. Of 28 TME cytokines and growth factors tested, we identified Oncostatin M (OSM) as the most potent inducer of mesenchymal/CSC properties. OSM-induced plasticity was Signal Transducer and Activator of Transcription 3 (STAT3)-dependent, and also required a novel intersection with transforming growth factor-ß (TGF-ß)/SMAD signaling. OSM/STAT3 activation promoted SMAD3 nuclear accumulation, DNA binding and induced SMAD3-dependent transcriptional activity. Suppression of TGF-ß receptor activity or ablation of SMAD3 or SMAD4, but not SMAD2, strongly suppressed OSM/STAT3-mediated plasticity. Moreover, removal of OSM or inhibition of STAT3 or SMAD3 resulted in a marked reversion to a non-invasive, epithelial phenotype. We propose that targeted blockade of the STAT3/SMAD3 axis in tumor cells may represent a novel therapeutic approach to prevent the plasticity required for metastatic progression and tumor recurrence.

Identification of genes encoding squalestatin S1 biosynthesis and in vitro production of new squalestatin analogues.

A gene cluster responsible for the biosynthesis of squalestatin S1 (SQS1, 1) was identified by full genome sequencing of two SQS1-producing ascomycetes: Phoma sp. C2932 and unidentified fungus MF5453. A transformation protocol was established and a subsequent knockout of one PKS gene from the cluster led to loss of SQS1 production and enhanced concentration of an SQS1 precursor. An acyltransferase gene from the cluster was expressed in E. coli and the expressed protein MfM4 shown to be responsible for loading acyl groups from CoA onto the squalestatin core as the final step of biosynthesis. MfM4 appears to have a broad substrate selectivity for its acyl CoA substrate, allowing the in vitro synthesis of novel squalestatins.

Expression of steroid hormone receptors in the genital structures of a true hermaphrodite pug dog.

Hermaphroditism is a rare and a not well-understood disordered sexual development (DSD) in dogs. The objective of the study was to analyse the sex steroid hormone receptor (STHR) expression patterns in the internal genital structures, because the responsiveness of the different tissue types to the steroid hormones may have a key role in pathological alterations based on DSDs. Furthermore, the adhesion molecule ß-catenin was investigated by means of immunohistochemistry because of its important role in development, tissue integrity and disease. Molecular sexing was performed via PCR targeting DBX/DBY genes to identify the pug dog as a true XX hermaphrodite. The portions of uterine tissue revealed comparable expression patterns for STHRs as investigated in normal female reproductive tissue. In the male parts, ß-catenin showed strong expression in the Sertoli cells of the seminiferous tubules; this was in contrast to normal testicular tissue. Likewise, the layers of smooth muscle actin-positive cells surrounding the seminiferous tubules were reduced in the hermaphrodite. The results of this study deepen the knowledge of tissue characteristics in a hermaphrodite dog and highlight the importance of early diagnosis because the STH responsiveness in maldeveloped reproductive tissue might lead to serious problems for the dog.

Measurement of the branching fraction B(Λc+ → pK-π+).

We present the first model-independent measurement of the absolute branching fraction of the Λ(c)(+) → pK(-)π(+) decay using a data sample of 978 fb(-1) collected with the Belle detector at the KEKB asymmetric-energy e(+)e(-) collider. The number of Λ(c)(+) baryons is determined by reconstructing the recoiling D((*)-) pπ(+) system in events of the type e(+)e(-) → D((*)-) pπ(+)Λ(c)(+). The branching fraction is measured to be B(Λ(c)(+) → pK(-)π(+)) = (6.84 ± 0.24(-0.27)(+0.21))%, where the first and second uncertainties are statistical and systematic, respectively.

Characterization of foamy epithelial surface cells in the canine endometrium.

In mature bitches, endometrial epithelial surface cells modify function and corresponding morphology during the oestrous cycle. During late metoestrous, endometrial epithelial surface cells frequently accumulate fat and thereby adopt a foamy morphology. This cyclic appearance of foamy endometrial epithelial cells (fEECs) seems to be physiological in the dog, whereas in other species, it indicates pathological changes. Function of these fEECs has not been identified until now. Therefore, the aim of the study was to characterize the fEECs by means of transmission electron microscopy and immunohistochemistry. Different manifestations of fEECs were observed and analysed with regard to proliferative activity and presence of different epithelial adhesion molecules including PLEKHA7, ß-catenin and E-cadherin. PLEKHA7 was restricted to the apical regions of the fEECs, whereas E-cadherin and ß-catenin were demonstrated basolateral. The immunohistochemical detection of steroid hormone receptors demonstrated the responsiveness of the fEECs to steroid hormones. Intense progesterone receptor expression was observed in the fEECs indicating a high responsiveness to this hormone. Considering a potential function of the fEECs, we hypothesized that leptin, a hormone produced by other lipid-accumulating cells and described to be involved in reproduction, in particular during implantation, might also originate from the fEECs which was confirmed by immunohistochemical methods. Moreover, leptin receptor was found in fEECs indicating the fEECs as both, source and target for leptin. Therefore, we conclude that fEECs in the canine uterus have a potential role in early pregnancy events and that the different observed manifestations might simply reflect the variations of signs of pseudopregnancy among bitches.

Hyperactivation of EGFR and downstream effector phospholipase D1 by oncogenic FAM83B.

Despite the progress made in targeted anticancer therapies in recent years, challenges remain. The identification of new potential targets will ensure that the arsenal of cancer therapies continues to expand. FAM83B was recently discovered in a forward genetic screen for novel oncogenes that drive human mammary epithelial cell (HMEC) transformation. We report here that elevated FAM83B expression increases Phospholipase D (PLD) activity, and that suppression of PLD1 activity prevents FAM83B-mediated transformation. The increased PLD activity is engaged by hyperactivation of epidermal growth factor receptor (EGFR), which is regulated by an interaction involving FAM83B and EGFR. Preventing the FAM83B/EGFR interaction by site-directed mutation of lysine 230 of FAM83B suppressed PLD activity and MAPK signaling. Furthermore, ablation of FAM83B expression from breast cancer cells inhibited EGFR phosphorylation and suppressed cell proliferation. We propose that understanding the mechanism of FAM83B-mediated transformation will provide a foundation for future therapies aimed at targeting its function as an intermediary in EGFR, MAPK and mTOR activation.

Evaluation of Xist expression in preattachment equine embryos.

Until now, sex determination in equine embryos has been performed by detection of Y-chromosome-specific sequences only. In the present study, expression of a Barr-body-specific marker, the X-inactivated-specific transcript (Xist) gene, whose gene product consists of RNA which coats and thereby inactivates one of the X chromosomes, was investigated in equine embryos produced in vivo. Preattachment embryos at different times after ovulation (Day 8: n = 9; Day 10: n = 12; Day 12: n = 15) were analyzed for Xist RNA expression using quantitative and qualitative reverse transcription-polymerase chain reaction (RT-PCR). Female and male primary equine dermal cell cultures were used as positive and negative controls, respectively. Embryos tested negative for Xist were evaluated for expression of the male-specific eSRY gene by qualitative PCR at the DNA level. From 36 embryos assessed by qualitative RT-PCR, 18 showed positive Xist expression (50%). From 29 embryos tested by quantitative RT-PCR, 16 showed positive Xist expression (55%). All of the Xist-negative equine embryos tested by quantitative PCR were positive for eSRY. We also demonstrated by strand-specific RT-PCR that in the horse, as in humans, the counter transcript Tsix seems to be truncated not reaching Exon 1. In contrast to many other species, neither Xist nor Tsix was expressed in equine male testicular tissue. The results demonstrate that expression of Xist is restricted to female equine embryos. Xist can thus be considered an X-inactivation-specific marker which can be used in concert with Y-specific markers for sex determination.

Sex-specific effects of spironolactone on blood pressure in gonadectomized male and female Wistar rats.

A low-salt diet is known to decrease and salt excess to increase blood pressure in humans and rodents. Sex steroids seem to play a role in salt dependent hypertension. However, little is known about sex differences in mineralocorticoid receptor blockade between male and female rats. The objective of the work was at first to investigate the effects of a low-salt vs. a high-salt diet on blood pressure without the influence of gonadal steroids in male and female rats. Second, to determine the sex-specific effects of mineralocorticoid receptor blockade by spironolactone in high-salt and low-salt fed gonadectomized male and female animals. Normotensive male and female Wistar rats were gonadectomized and put on a low (NaCl<0.03%) or high (NaCl=4%) salt diet. On each diet animals received spironolactone or placebo. Blood pressure was measured by tail-cuff-method; 24-h urine samples were collected in metabolic cages and blood was collected for hormonal measurements. High-salt diet significantly increased systolic blood pressure in both sexes. This effect could be blocked effectively by spironolactone only in male rats. Spironolactone treatment significantly increased aldosterone levels in males and females independent of the sodium content of the diet. High sodium diet significantly increased relative kidney weight, which was not altered by spironolactone treatment. Independently of gonadal steroids a high-salt diet increased blood pressure in gonadectomized male and female rats. Spironolactone lowered blood pressure only in male not in female rats on a high-salt diet clearly indicating sex-specific effects of the mineralo-corticoid antagonist spironolactone.

Ectopic endometrial tissue in mesonephric duct remnants in bitches.

Common congenital embryonic remnants of the canine female genital tract are Gartner cysts originating from mesonephric duct remnants. They can increase in size and lead to adverse effects in fertility and health. In the present study, three cases of mesonephric remnants in bitches were analysed. The mesonephric remnants featured an inner lining endometrium comprising surface epithelium, glands and stroma. This ectopic endometrium was further characterized by immunohistochemistry (oestrogen and progesterone receptors, proliferation activity, cytokeratin, alpha smooth muscle actin, and vimentin) and lectin histochemistry compared with normal uterine endometrium. Furthermore, hypertrophic cells at the serosal site of the uteri were detected and analysed in the same way compared with normal serosa. The ectopic endometrium of case no. 2 mesonephric remnant was comparable with normal endometrium whereas in nos 1 and 3 uteri the ectopic endometrium was reduced in thickness. In all mesonephric remnants, surface and glandular epithelial cells of the ectopic endometrium gave positive immunoreactions for cytokeratin, oestrogen and progesterone receptors and showed lectin-binding patterns comparable with normal endometrium. Some of the stromal cells of the ectopic endometria were smooth muscle actin and vimentin positive. Mitotic activity of the ectopic endometria was comparable with normal endometria. Hypertrophic epithelial cells of the serosal side showed positive reactions to anti-oestrogen receptor and anti-cytokeratin immunohistochemistry as well as lectin binding patterns and mitotic activity comparable with the normal canine serosa. The present study is the first considerable immunohistochemical characterization of canine mesonephric remnants and discusses the appearance of ectopic endometrium in mesonephric remnants.

Alpha-lipoic acid inhibits thrombin-induced fetal membrane weakening in vitro.

Cytokine-mediated inflammation and abruption-induced thrombin generation are separately implicated in matrix metalloproteinase (MMP)-mediated weakening of fetal membranes (FM) leading to preterm premature rupture of the fetal membranes (PPROM). At term, FM of both labored vaginal and unlabored Cesarean deliveries exhibit a weak zone overlying the cervix exhibiting ECM remodeling characterized by increased MMP9 protein and activity. We have reproduced these biochemical changes as well as FM weakening in vitro using tumor necrosis factor-alpha (TNF) and interleukin (IL)-1ß, inflammatory cytokines implicated in PPROM. Additionally, we have reported that the antioxidant and NFκB inhibitor alpha-lipoic Acid (LA) blocks these TNF-induced effects. We now present the first direct evidence that thrombin also can induce FM weakening in vitro, and LA treatment inhibits this thrombin-induced-weakening. Full thickness FM fragments from unlabored Cesarean deliveries were incubated with increasing doses of thrombin (0-100 u/ml) for 48 h. Fragments were then strength tested (breaking force and work to rupture) using our published methodology. MMP3 and 9 levels in tissue extracts were determined by Western blot and densitometry. To determine the effect of LA, FM fragments were incubated with control medium or 10 u/ml thrombin, with or without 0.25 mM LA. Strength testing and MMP induction were determined. Thrombin induced a dose-dependent decrease in FM strength (42% baseline rupture force and 45% work to rupture) coupled with a dose-dependent increase in MMP3 and 9 expression (all p < 0.001). Treatment of FM with 0.25 mM LA completely inhibited thrombin-induced FM weakening and MMP expression (all p < 0.001). Thrombin treatment of cultured FM induces mechanical weakening and increased MMP3 and 9. Treatment of FM with LA inhibits these thrombin-induced effects. We speculate LA may prove clinically useful in prevention of PPROM associated with abruption.

Pseudo-placentational endometrial cysts in a bitch.

Cystic alterations of the canine endometrium compromise reproduction and fertility of the bitch and may lead to life-threatening diseases, such as pyometra. Even without clinical evidence, reduction of the uterine lumen by cysts implicates disturbances during migration, nidation and development of the embryo. Several studies point to the high variability of morphology of uterine endometrial cysts but they lack detailed analyses of alterations. In the present study, immunohistochemistry was used to investigate the expression of steroid hormone receptors (oestrogen, progesterone), proliferation activity, inflammation and infection in the cystic affected tissue regions in contrast to the normal endometrium. Oestrogen receptor expression showed a high density of receptors throughout the surface epithelial cells, crypt epithelial cells, glandular epithelial cells and stromal cells of the normal endometrium as well as the cystic affected regions. Proliferation in the cysts was verified in the middle and basal cells of the crypts. Neither in the endometrium nor in the cysts inflammatory processes or evidence of infection could be detected. Furthermore, lectin histochemistry and electron microscopic methods showed that lectin binding patterns and cell morphology of internal epithelial lining and surface epithelium of the cysts can be used to characterize and distinguish different types of cystic alterations. Analogies between epithelial cells of the glandular chambers of the canine placenta and the cystic cellular morphology, steroid hormone receptor distribution as well as lectin binding patterns of the endometrial cysts, as observed in this study, suggest to introduce the term 'pseudo-placentational endometrial cysts'.

Remission of a B cell CLL-associated membranoproliferative glomerulonephritis Type I with rituximab and bendamustine.

In a 56-year-old white male patient, a membranoproliferative glomerulonephritis Type I was diagnosed after a 12-month history of low grade B cell lymphoma (Binet A). HIV, Hepatitis B and C serology were negative. Due to an impairment of renal function despite chemotherapy with COP, an immunochemotherapy consisting of rituximab (6 cycles) and bendamustine (4 cycles) was given. This therapeutic approach caused a complete remission of the nephrotic syndrome. Renal function and arterial hypertension improved markedly. In addition, urinary sediment became normal and proteinuria disappeared completely.

A phase IB, open-label dose-escalating study of the oral angiogenesis inhibitor PTK787/ZK 222584 (PTK/ZK), in combination with FOLFOX4 chemotherapy in patients with advanced colorectal cancer.

BACKGROUND: This open-label, phase IB study was undertaken to determine the safety/toxicity profile and recommended dose of oral once-daily PTK787/ZK 222584 (PTK/ZK) combined with oxaliplatin/5-fluorouracil (5-FU)/leucovorin (FOLFOX4) chemotherapy in patients with advanced colorectal cancer. Secondary objectives were to assess full pharmacokinetics and gather preliminary evidence of antitumor activity. PATIENTS AND METHODS: Thirty-five patients received escalating doses of PTK/ZK (range 500-2000 mg daily) continuously. Concurrent FOLFOX4 chemotherapy was administered on days 1 and 2 and repeated every 14 days. Dose escalation of PTK/ZK was continued until maximum tolerated dose (MTD) was established and additional patients were then enrolled at MTD dosage. RESULTS: Mean treatment duration of PTK/ZK was 9.5 months. The MTD was 1250 mg daily with dizziness being the most frequent dose-limiting toxicity (DLT). Hypertension (23%, grade 3) and neutropenia (37%, grades 3 + 4) were the most frequent grade 3 or 4 adverse events. Pharmacokinetic analyses found no evidence for interactions between PTK/ZK and the combination of 5-FU, leucovorin, and oxaliplatin during concomitant use. Median progression-free survival was 11.4 months. CONCLUSION: The MTD of PTK/ZK in combination with FOLFOX4 in this patient population is 1250 mg daily. The combination is feasible and safe and is not associated with significant pharmacokinetic interactions.

Beyond mammography screening: quality assurance in breast cancer diagnosis (The QuaMaDi Project).

As many breast cancer cases are detected outside mammographic screening, a multidisciplinary quality management (QuaMaDi) project involving gynaecologists, double reading by radiologists. and centralised assessment, documentation, evaluation and feedback was implemented into routine breast cancer diagnosis in part of Schleswig-Holstein (Germany) with a population of 365,000 women. A cohort of 59,514 patients eligible for diagnostic mammography was examined from May 2001 to December 2005 and quality indicators, breast cancer incidence and tumour stage distribution were analysed. A total of 102,744 diagnostic processes were initiated, for 23.8% of which (24,470) a third expert reading at the reference centre was performed. Further assessment was recommended for 6.3% (6442) of all patients. In total, 1056 breast cancer cases were diagnosed (10.3 per 1000 examinations). Patients of the QuaMaDi project had a higher proportion of 'in situ' and T1 tumours (62.6% vs Schleswig-Holstein: 48.6%), showing that the implementation of high standards in routine diagnostic mammography can improve the quality of breast cancer diagnosis and care.

[Problems in defining obesity in prepubescent children: consequences for assessing the requirements for medical rehabilitation]. / Probleme der Definition der Adipositas bei Kindern vor der Pubertät: Konsequenzen für die Ermittlung ihres Rehabilitationsbedarfs.

INTRODUCTION: Prevalence of obesity has been on the increase in recent years. In contrast to obesity in adulthood, childhood obesity is still not uniformly defined. This results in problems to determine the need for medical rehabilitation. METHODS: The Kiel Obesity Prevention Study (KOPS) is a cross-sectional study assessing the nutritional status of 5-7 year-old German children. Three different definitions of overweight and obesity (German reference data for triceps skinfold thickness [90(th) percentile] and BMI [90(th)/97(th) percentile] as well as an international standard for BMI [extrapolated to levels of adults]) were applied to 1,643 children of KOPS enrolled between 1996 and 2000 (19 % of all first-graders in Kiel in this period). RESULTS: The prevalence of overweight varies from 9 to 21 % depending on the applied definition. With the definitions of overweight and obesity based on newer BMI percentiles a part of overweight children are not classified as such. The present state of art is that there is only a need for obese children for medical rehabilitation: these are 3.3 and 3.5 % of 5-7 year-old children in Kiel, respectively. CONCLUSIONS: Experts should work out an agreement concerning a uniform definition of childhood obesity. Currently, medical rehabilitation services are offered only to extremely obese children. There is a need for more and earlier preventive measures.

Syntheses of halogenated ethenyl isocyanide chromium complexes as organometallic precursor molecules for ethenyl and ethynyl isocyanides.

The radical alkylation of tetraethylammonium pentacarbonyl(cyano)chromate 1 yielded the halogenated ethyl isocyanide complexes [(CO)5Cr(CN-CClX-CClYF)] 3 (a, X= Cl, Y= F; b, X = F, Y= F and c, X=Y= Cl). Dehalogenation of 3 using zinc in diethyl ether gave [(CO)5Cr(CN-CX=CFY)] 4. The compounds 4a, b reacted with various nucleophiles exclusively at the difluoromethylene group. The unstable phosphorane 5, which is formed on reaction of 4b with trimethylphosphane, decomposed thermally and on hydrolysis yielding pentacarbonyl(1,2-difluoroethenyl isocyanide)chromium (6). The cyano substituent can be introduced in the beta position of the isocyanide function by reaction of 4a, b with potassium cyanide, leading to the formation of [(CO)5Cr(CN-CX=CF-CN)] (7). Reactions of 4a, b with organolithium or organomagnesium compounds yielded [(CO)5Cr(CN-CX=CF-R)] (8) and [(CO)5Cr(CN-CF=CF-C...C-CF=CF-NC)Cr(CO)5] (10). The trimethylsilyl group in 8a, b, d could be removed by a solution of potassium carbonate in methanol leading to [(CO)5Cr(CN-CX=CF-Cn-H)] (11) (n=2,4). Octacarbonyldicobalt reacted with 8e under coordination of the C-C triple bond to the hexacarbonyldicobalt fragment, resulting in the cluster compound 12. The crystal and molecular structure of 8i, 11 a, b, and 12 were elucidated by X-ray crystallography. The alkenyl and alkynyl isocyanides CN-CCl=CF2 (13a), CN-CF=CF2 (13b), CN-CCl=CClF (13c), CN-CF=CFH (14), CN-CC-H (15), CN-CC-CN (16), and CN-CCl=CF-CN (17) were obtained by flash vacuum pyrolysis of 4a, 4b, 4c, 6, and 7a, respectively.

Isotretinoin use in acne: prospective evaluation of adverse events.

BACKGROUND: Isotretinoin is an effective treatment for severe acne. Although the spectrum of side effects has been well documented, the changing incidence of such side effects over the course of treatment has not been studied in detail. OBJECTIVES: The purpose of our study was to examine a group of patients monthly over their course of treatment and prospectively document the side effects experienced. METHODS: Over the period between January 1991 and July 1996, 124 courses of treatment with isotretinoin for severe acne were followed. The patients were treated for 4 months at a dose of 1 mg per kg body weight. A questionnaire was administered monthly, inquiring specifically about side effects known to be associated with isotretinoin. Any additional side effects were also noted. RESULTS: The majority of patients experienced persistent dryness of lips. Dry eyes affected 40% of patients; this continued throughout treatment in 25%. Contact lens wearers were more likely to develop conjunctivitis. Lower back pain was reported early in about 30% of patients and fewer than 10% of patients would develop it later in the course of treatment. Arthralgia was noted in 16.5% of patients at the first visit and there was little change with ongoing treatment. Hair loss was experienced in a small percentage but was rarely noted on more than one occasion. Headaches occurred in less than 10% and were occasionally severe, but most often intermittent and recorded at a single visit. Depression occurred in 4% of patients and tended to persist throughout the treatment. All these patients completed the full course of treatment. CONCLUSION: This prospective analysis has shown that patients treated with isotretinoin experienced a predictable series of side effects. Some occurred fleetingly, but several persisted for the duration of treatment.