Cellular Pre-Adaptation to the High O2 Concentration Used in Standard Cell Culture Confers Resistance to Subsequent H2O2-Induced Cell Death.

The addition of hydrogen peroxide (H2O2) to cultured cells is widely used as a method to modulate redox-regulated cellular pathways, including the induction of programmed cell death in cell culture experiments and the testing of pro- and antioxidant compounds. Here, we assessed the effect on the cellular response to H2O2 of pre-adapting squamous cell carcinoma cells (A431) to the standard cell culture oxygenation of 18.6% O2, compared to cells pre-adapted to a physiological skin O2 concentration (3.0% O2). We showed that cells pre-adapted to 18.6% O2 resisted H2O2-induced cell death compared to cells pre-adapted to 3.0% O2 for 96 h prior to treatment with H2O2. Moreover, the enzymatic activities of catalase and glutathione reductase, as well as the protein expression levels of catalase, were higher in cells pre-adapted to 18.6% O2 compared to cells pre-adapted to 3.0% O2. H2O2-resistant cells, pre-adapted to 18.6% O2, exhibited increased nuclear Nrf-2 levels. It is concluded that A431 cells pre-adapted to standard cell culture oxygenation conditions resist H2O2-induced cell death. This effect may be related to their heightened activation of Nrf-2.

Aural health awareness and incident prevention in UK scuba divers.

INTRODUCTION: Otological disorders, including Eustachian tube dysfunction (ETD), are commonly observed in divers. Data were gathered to observe the prevalence of ear disorders, and awareness of ear health recommendations for recreational divers in the United Kingdom. METHODS: An anonymous online survey included: diver/diving demographics, the validated Eustachian Tube Dysfunction Questionnaire 7 (ETDQ-7) (a mean score of ≥ 2.1 indicating the presence of dysfunction), pre-existing ear health conditions, medications, decongestants and knowledge of diving and ear health guidance. RESULTS: A total of 790 divers (64% males) responded (age range 16-80, median 47 years). An ETDQ-7 mean score of ≥ 2.1 was calculated in 315 of 790 respondents (40%), indicating varying degrees of ETD; 56/315 (18%) recorded a pre-existing ear condition. Ear disorders, (external, middle, and inner ear issues) since learning to dive were recorded by 628/790 (79%) of respondents; 291/628 (46%) did not seek medical advice. ETDQ-7 scores of ≥ 2.1 to 6.6 were reported by 293/628 (47%). Six reported inner ear decompression sickness. Decongestants were used by 183/790 (23%). Two hundred and seventy-seven of 790 divers (35%) had aborted a dive with ear problems. Only 214/790 (27%) of respondents were aware of the United Kingdom Diving Medical Committee guidance regarding ear health and diving. CONCLUSIONS: Ear problems and ETD since diving were widely reported in this cohort of divers, with not all divers in this study aware of ear health recommendations and advice.

Flying after diving: a questionnaire-based evaluation of pre-flight diving behaviour in a recreational diving cohort.

INTRODUCTION: Divers are recommended to observe a pre-flight surface interval (PFSI) ≥ 24 hours before boarding a plane following a diving vacation. Decompression sickness (DCS) symptoms may occur during or post-flight. This study aimed to examine the adherence of PFSI ≥ 24 in vacationing divers, and if any perceived signs and symptoms of DCS during or after flight were experienced. METHODS: An anonymous online survey was publicised through diving exhibitions and social media. Data included diver/diving demographics, PFSI before flight, flight details, and perceived signs and symptoms of DCS during or after flight. RESULTS: Data from 316 divers were examined (31% female) with the age range 17-75 years (median 49). Divers recorded 4,356 dives in the week preceding the flight, range 1-36 (median 14). Overall, 251/316 (79%) respondents reported a PFSI of ≥ 24 hours. PFSIs of < 12 hours were reported by 6 respondents. Diagnosed and treated DCS developing during, and post flight was reported by 4 divers with PFSIs ≥ 24 hours and by 2 divers with PFSIs < 24 hours. Fifteen divers boarded a plane with perceived symptoms of DCS. CONCLUSIONS: These data suggest that most divers in this study observed the recommendations of a ≥ 24 hour PFSI with safe outcomes.

Diving and mental health: the potential benefits and risks from a survey of recreational scuba divers.

INTRODUCTION: Scuba diving is physically and cognitively demanding. Medical guidance regarding physical and mental health (MH) issues and related prescribed medication is often based on limited evidence. There is a paucity of data concerning diving with MH issues. This survey aimed to investigate the prevalence of MH issues and use of prescription medications among United Kingdom (UK) sport divers, and the rate of non-compliance with current guidance among divers suffering depression and anxiety. The positive effects of scuba diving on MH were also considered. METHODS: An anonymous online survey was publicised through diving exhibitions and social media. Measures included diver and diving demographics; GAD-7 Anxiety and PHQ-9 depression questionnaires; diagnosed current and/or past MH conditions; medication usage; comorbid medical conditions/treatments; disclosure of past/current MH issues; and perceived MH benefits of diving. RESULTS: Data from 729 respondents revealed MH issues at rates comparable with the general population. Current and/or past MH issues were reported by 111/729, with 60 having active diagnoses, and 45/60 taking prescribed psychotropic medications; 21/45 did not declare their medication on diver self-certification medical forms. The activity of diving was thought to be beneficial to MH by 119/729 respondents. CONCLUSIONS: Divers experienced expected levels of MH issues, but did not comply with current medical guidelines on modifying or abstaining from diving activity or reporting their MH condition. Changes may be needed to diver training to encourage more accurate reporting and aid development of evidence-based protocols. Guidelines could be reconsidered in light of current diver behaviour, risks and potential MH benefits.

Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia.

In vivo, mammalian cells reside in an environment of 0.5-10% O2 (depending on the tissue location within the body), whilst standard in vitro cell culture is carried out under room air. Little is known about the effects of this hyperoxic environment on treatment-induced oxidative stress, relative to a physiological oxygen environment. In the present study we investigated the effects of long-term culture under hyperoxia (air) on photodynamic treatment. Upon photodynamic irradiation, cells which had been cultured long-term under hyperoxia generated higher concentrations of mitochondrial reactive oxygen species, compared with cells in a physioxic (2% O2) environment. However, there was no significant difference in viability between hyperoxic and physioxic cells. The expression of genes encoding key redox homeostasis proteins and the activity of key antioxidant enzymes was significantly higher after the long-term culture of hyperoxic cells compared with physioxic cells. The induction of antioxidant genes and increased antioxidant enzyme activity appear to contribute to the development of a phenotype that is resistant to oxidative stress-induced cellular damage and death when using standard cell culture conditions. The results from experiments using selective inhibitors suggested that the thioredoxin antioxidant system contributes to this phenotype. To avoid artefactual results, in vitro cellular responses should be studied in mammalian cells that have been cultured under physioxia. This investigation provides new insights into the effects of physioxic cell culture on a model of a clinically relevant photodynamic treatment and the associated cellular pathways.

Manipulation of Oxygen and Endoplasmic Reticulum Stress Factors as Possible Interventions for Treatment of Multiple Sclerosis: Evidence for and Against.

Multiple sclerosis (MS) is normally considered a chronic inflammatory disease of the central nervous system (CNS), where T-cells breaching the blood brain barrier react against proteins of the axonal myelin sheaths, leading to focal plaques and demyelination in the brain and spinal cord. Many current therapies are immunosuppressive in nature and are designed to target the immune system at an early stage of the disease. But there is no cure and MS may evolve into a neurodegenerative disease, where immunomodulatory treatments appear less effective. Neurodegeneration is influenced by oxidative and endoplasmic reticulum (ER) mediated stress which can be induced independently of immune processes. Since 1970, MS patients have been self-managing their long term symptoms using hyperbaric oxygen and reporting improvement in their symptoms, especially bladder control. In contrast, the majority of clinical trial evidence does not support the views of patients. Therefore does oxygen under pressure affect brain tissue by modulating oxidative or ER stress at the cellular level resulting in CNS tissue repair or deterioration? This chapter reviews our understanding and the role of oxidative and ER stress in the context of employing hyperoxia treatments to treat MS and evaluate its effects on neural cells.

The hydroxypyridinone iron chelator CP94 increases methyl-aminolevulinate-based photodynamic cell killing by increasing the generation of reactive oxygen species.

Methyl-aminolevulinate-based photodynamic therapy (MAL-PDT) is utilised clinically for the treatment of non-melanoma skin cancers and pre-cancers and the hydroxypyridinone iron chelator, CP94, has successfully been demonstrated to increase MAL-PDT efficacy in an initial clinical pilot study. However, the biochemical and photochemical processes leading to CP94-enhanced photodynamic cell death, beyond the well-documented increases in accumulation of the photosensitiser protoporphyrin IX (PpIX), have not yet been fully elucidated. This investigation demonstrated that MAL-based photodynamic cell killing of cultured human squamous carcinoma cells (A431) occurred in a predominantly necrotic manner following the generation of singlet oxygen and ROS. Augmenting MAL-based photodynamic cell killing with CP94 co-treatment resulted in increased PpIX accumulation, MitoSOX-detectable ROS generation (probably of mitochondrial origin) and necrotic cell death, but did not affect singlet oxygen generation. We also report (to our knowledge, for the first time) the detection of intracellular PpIX-generated singlet oxygen in whole cells via electron paramagnetic resonance spectroscopy in conjunction with a spin trap.

Hyperbaric oxygen for patients with chronic bowel dysfunction after pelvic radiotherapy (HOT2): a randomised, double-blind, sham-controlled phase 3 trial.

BACKGROUND: Hyperbaric oxygen has been used as a therapy for patients experiencing chronic intestinal syndromes after pelvic radiotherapy for decades, yet the evidence to support the use of this therapy is based almost exclusively on non-randomised studies. We aimed to provide conclusive results for the clinical benefits of hyperbaric oxygen in patients with chronic bowel dysfunction after radiotherapy for pelvic malignancies. METHODS: HOT2 was a double-blind, sham-controlled, phase 3 randomised study of patients (≥18 years) with chronic gastrointestinal symptoms for 12 months or more after radiotherapy and which persisted despite at least 3 months of optimal medical therapy and no evidence of cancer recurrence. Participants were stratified by participating hyperbaric centre and randomly assigned (2:1) by a computer-generated list (block size nine or 12) to receive treatment with hyperbaric oxygen therapy or sham. Participants in the active treatment group breathed 100% oxygen at 2·4 atmospheres of absolute pressure (ATA) and the control group breathed 21% oxygen at 1·3 ATA; both treatment groups received 90-min air pressure exposures once daily for 5 days per week for a total of 8 weeks (total of 40 exposures). Staff at the participating hyperbaric medicine facilities knew the allocated treatment, but patients, clinicians, nurse practitioners, and other health-care professionals associated with patients' care were masked to treatment allocation. Primary endpoints were changes in the bowel component of the modified Inflammatory Bowel Disease Questionnaire (IBDQ) score and the IBDQ rectal bleeding score 12 months after start of treatment relative to baseline. The primary outcome was analysed in a modified intention-to-treat population, excluding patients who did not provide IBDQ scores within a predetermined time-frame. All patients have completed 12 months of follow-up and the final analysis is complete. The trial is registered with the ISRCTN registry, number ISRCTN86894066. FINDINGS: Between Aug 14, 2009, and Oct 23, 2012, 84 participants were randomly assigned: 55 to hyperbaric oxygen and 29 to sham control. 75 (89%) participants received 40 pressure exposures, all participants returned the IBDQ at baseline, 75 (89%) participants returned the IBDQ at 2 weeks post-treatment, and 79 (94%) participants returned the IBDQ at 12 months post-start of treatment. Patients were excluded from analyses of co-primary endpoints if they had missing IBDQ scores for intestinal function or rectal bleeding at baseline or at 12 months. In an analysis of 46 participants in the active treatment group and 23 participants in the control group, we found no significant differences in the change of IBDQ bowel component score (median change from baseline to 12 months of 4 (IQR -3 to 11) in the treatment group vs 4 (-6 to 9) in the sham group; Mann-Whitney U score 0·67, p=0·50). In an analysis of 29 participants in the active treatment group and 11 participants in the sham group with rectal bleeding at baseline, we also found no significant differences in the change of IBDQ rectal bleeding score (median change from baseline to 12 months of 3 [1 to 3] in the treatment group vs 1 [1 to 2] in the sham group; U score 1·69, p=0·092). Common adverse events in both groups were eye refractive changes (three [11%] of 28 patients in the control group vs 16 [30%] of 53 patients in the treatment group), increased fatigue (three [11%] vs two [4%]), and ear pain (six [21%] vs 15 [28%]). Eight serious adverse events were reported in eight patients: two were reported in two patients in the control group (tonsillitis requiring surgery [grade 3]; recurrent cancer of the vulva [grade 4]) and six serious adverse events were reported in six patients in the treatment group (malignant spinal cord compression requiring surgery [grade 3]; malignant paraortic lymph node involvement requiring surgery [grade 3]; recurrence of vomiting and dehydration [grade 3]; diarrhoea and fever associated with Campylobacter infection [grade 3]; recurrence of abdominal pain, bloating, diarrhoea, and urinary tract infection [grade 3]; aneurysm [grade 4]), none of which were deemed treatment-related. INTERPRETATION: We found no evidence that patients with radiation-induced chronic gastrointestinal symptoms, including those patients with rectal bleeding, benefit from hyperbaric oxygen therapy. These findings contrast with evidence used to justify current practices, and more level 1 evidence is urgently needed. FUNDING: Cancer Research UK and National Health Service (NHS) funding to the National Institute of Health Research Biomedical Research Centre at The Royal Marsden and the Institute of Cancer Research.

Does self-certification reflect the cardiac health of UK sport divers?

BACKGROUND: Since 2009, the United Kingdom diving incident data show an increasing number of fatalities in the over-50s age group. Previous studies also suggest some divers take cardiac medications. Since 2001, diving medicals have not been mandatory for UK sport divers. Instead, an annual medical self-certification form, submitted to their club/school or training establishment, is required. We documented in a survey of UK sport divers the prevalence of cardiac events and medications and the frequency of medical certifications. METHODS: An anonymous on-line questionnaire was publicised. Measures included diver and diving demographics, prescribed medications, diagnosed hypertension, cardiac issues, events and procedures, other health issues, year of last diving medical, diagnosed persistent foramen ovale (PFO), smoking and alcohol habits, exercise and body mass index. RESULTS: Of 672 completed surveys, hypertension was reported by 119 (18%) with 25 of these (21%) having not had a diving medical. Myocardial infarction 6 (1%), coronary artery bypass grafting 3 (< 1%), atrial fibrillation 19 (3%) and angina 12 (2%) were also reported. PFOs were reported by 28 (4%), with 20 of these opting for a closure procedure. From 83 treated incidences of decompression illness (DCI), 19 divers reported that a PFO was diagnosed. CONCLUSIONS: Divers inevitably develop health problems. Some continue to dive with cardiac issues, failing to seek specialised diving advice or fully understand the role of the diving medical. Physicians without appropriate training in diving medicine may inform a diver they are safe to continue diving with their condition without appreciating the potential risks. The current procedure for medical screening for fitness to dive may not be adequate for all divers.

Hyperbaric oxygen therapy accelerates osteoblast differentiation and promotes bone formation.

OBJECTIVES: Hyperbaric oxygen therapy (HBO) has been used as an adjunctive therapy in the treatment of radiotherapy or bisphosphonate-induced osteonecrosis of the jaw however the effect of HBO on osteoblast formation and mineralisation has not been extensively studied. The current study therefore examined the effects of HBO, elevated pressure or elevated oxygen alone on osteoblast differentiation and bone nodule formation. METHODS: Saos-2 human osteoblast cells were exposed to HBO (2.4 ATA, 97.9% O2, 90 min per day), elevated pressure alone (2.4 ATA, 8.8% O2, 90 min per day) or elevated oxygen alone (1 ATA, 95% O2, 90 min per day) after culturing under normoxic or hypoxic conditions and osteoblast differentiation and bone formation assessed by alkaline phosphatase activity and calcein incorporation. Expression of key regulators of osteoblast differentiation and bone matrix proteins were assessed by quantitative PCR. RESULTS: Daily exposure to HBO accelerated the rate of osteoblast differentiation as determined by increased alkaline phosphatase activity and expression of type I collagen and Runx-2 mRNA during the early stages of culture. HBO also augmented bone nodule formation in hypoxic conditions. HBO had a more pronounced effect on these key markers of osteoblast differentiation than elevated oxygen or pressure alone. CONCLUSIONS: The data from this study shows that daily HBO treatment accelerated the rate of osteoblast differentiation leading to an increase in bone formation. CLINICAL SIGNIFICANCE: These studies add to our understanding of HBO's reparative action in osteonecrotic bone loss. In addition to stimulating angiogenesis HBO may also improve surgical outcomes through a direct beneficial effect on osteoblast differentiation generating a larger bone mass available for reconstruction.

Hyperbaric oxygen treatment reduces neutrophil-endothelial adhesion in chronic wound conditions through S-nitrosation.

Hyperbaric oxygen (HBO) therapy is an effective treatment for diabetic chronic wounds. HBO reduces inflammation and accelerates wound healing, by mechanisms that remain unclear. Here we examined a mechanism by which HBO may reduce neutrophil recruitment, through changes in endothelial and neutrophil adhesion molecule expression and function. Human umbilical vein endothelial cells and neutrophils were exposed to selected chronic wound conditions, comprising hypoxia in the presence of lipopolysaccharide and tumor necrosis factor-alpha, and then treated with HBO. We observed neutrophil adhesion to endothelial cells following treatment with chronic wound conditions, which was reversed by HBO treatment. This was partly explained by reduced expression of endothelial intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 by HBO. No changes in neutrophil adhesion molecule expression (CD18, CD11b, CD62L, CD31) were observed following HBO treatment. However, HBO decreased hydrogen peroxide generation by neutrophils, and induced nitrous oxide-related protein modifications. The transnitrosating agent S-nitroso-L-cysteine ethyl ester (600 µM) also reduced neutrophil adhesion to human umbilical vein endothelial cell monolayers, and the iNOS inhibitor 1400 W (10 µM) and HgCl2, which promotes the decomposition of S-nitrosothiols (1 mM), reversed the effect of HBO, suggesting that S-nitrosation may inhibit neutrophil-endothelial cell adhesion. This study indicates that HBO could reduce inflammation in wounds through reduced neutrophil recruitment, mediated by S-nitrosation.

Hyperbaric oxygen therapy suppresses osteoclast formation and bone resorption.

The cellular and molecular mechanism through which hyperbaric oxygen therapy (HBO) improves osteonecrosis (ON) is unclear. The present study therefore examined the effect of HBO, pressure and hyperoxia on RANKL-induced osteoclast formation in RAW 264.7 cells and human peripheral blood monocytes (PBMC). Daily exposure to HBO (2.4 ATA, 97% O2 , 90 min), hyperbaric pressure (2.4 ATA, 8.8% O2 , 90 min) or normobaric hyperoxia (1 ATA, 95% O2 , 90 min) significantly decreased RANKL-induced osteoclast formation and bone resorption in normoxic conditions. HBO had a more pronounced anti-osteoclastic effect than hyperoxia or pressure alone and also directly inhibited osteoclast formation and resorption in hypoxic conditions a hallmark of many osteolytic skeletal disorders. The suppressive action of HBO was at least in part mediated through a reduction in RANK, NFATc1, and Dc-STAMP expression and inhibition of hypoxia-induced HIF-1α mRNA and protein expression. This data provides mechanistic evidence supporting the use of HBO as an adjunctive therapy to prevent osteoclast formation and bone loss associated with low oxygen partial pressure.

Different oxygen treatment pressures alter inflammatory gene expression in human endothelial cells.

Hyperbaric oxygen has proven to be a useful treatment for chronic wounds. However, therapeutic conditions vary between treatment centers, and we wished to investigate the effects of different treatment pressures on cells under inflammatory conditions. Endothelial cells were exposed to a chronic wound model comprising hypoxia (2% O2 at 1 atmosphere absolute (atm abs); PO2 approximately 2 kPa) in the presence of 0.5 microg/ml lipopolysaccharide and 1 ng/ml TNF-alpha for 24 hours, then treated with normobaric oxygen (NBO2; 95%O2/5%CO2 at 1.0 atm abs; PO2 approximately 96.3 kPa), hyperbaric oxygen (HBO2) at 1.5 atm abs (1.5HBO2; 96.7%O2/3.3%CO2 at 1.5 atm abs; PO2 approximately 147 kPa), and HBO2 at 2.4 atm abs (2.4HBO2; 97.9%O2/2.1%CO2 at 2.4 atm abs; PO2 approximately 238 kPa). The mRNA expression of 92 inflammatory genes was then analyzed, and we identified changes in genes involved in adhesion molecule expression, angiogenesis and tissue remodeling, intracellular signaling, and cellular oxygen responses and redox signaling. We noted differences in expression between different treatment pressures, highlighting the need for further research into the use of different therapeutic protocols in the treatment of inflammatory conditions such as chronic wounds.

Changes in inflammatory gene expression induced by hyperbaric oxygen treatment in human endothelial cells under chronic wound conditions.

Hyperbaric oxygen (HBO) therapy involves the inhalation of 100% oxygen, whilst inside a chamber at greater than atmospheric pressure. It is an effective treatment for chronic diabetic wounds, although the molecular mechanisms involved remain unclear. We hypothesised that HBO could alter inflammatory gene expression in human endothelial cells via a reactive oxygen/nitrogen species-mediated pathway. Endothelial cells were exposed to a chronic wound model comprising hypoxia (2% O(2) at 1 atmosphere absolute (ATA); PO(2) ~2 kPa) in the presence of lipopolysaccharide and TNF-α for 24h, then treated with HBO for 90 min (97.5% O(2) at 2.4 ATA; PO(2) ~237 kPa). 5h post-HBO, 19 genes involved in adhesion, angiogenesis, inflammation and oxidative stress were downregulated. Notably, only angiogenin gene expression, which promotes both angiogenesis and nitric oxide production (reflected by increased eNOS protein expression in this study), was upregulated. This led to a decrease in endothelial IL-8 mRNA and protein, which could help alleviate inflammatory processes during chronic wound healing. This was no longer evident 22.5h post-HBO, demonstrating the importance of daily exposures in HBO treatment protocols. These studies indicate that elevated oxygen transiently regulates inflammatory gene expression in endothelial cells, which may enhance chronic wound healing.

Molecular diversity of fungal phylotypes co-amplified alongside nematodes from coastal and deep-sea marine environments.

Nematodes and fungi are both ubiquitous in marine environments, yet few studies have investigated relationships between these two groups. Microbial species share many well-documented interactions with both free-living and parasitic nematode species, and limited data from previous studies have suggested ecological associations between fungi and nematodes in benthic marine habitats. This study aimed to further document the taxonomy and distribution of fungal taxa often co-amplified from nematode specimens. A total of 15 fungal 18S rRNA phylotypes were isolated from nematode specimens representing both deep-sea and shallow water habitats; all fungal isolates displayed high pairwise sequence identities with published data in Genbank (99-100%) and unpublished high-throughput 454 environmental datasets (>95%). BLAST matches indicate marine fungal sequences amplified in this study broadly represent taxa within the phyla Ascomycota and Basidiomycota, and several phylotypes showed robust groupings with known taxa in phylogenetic topologies. In addition, some fungal phylotypes appeared to be present in disparate geographic habitats, suggesting cosmopolitan distributions or closely related species complexes in at least some marine fungi. The present study was only able to isolate fungal DNA from a restricted set of nematode taxa; further work is needed to fully investigate the taxonomic scope and function of nematode-fungal interactions.

Hyperbaric oxygen treatment induces platelet aggregation and protein release, without altering expression of activation molecules.

OBJECTIVES: To investigate the effect of hyperbaric oxygen (HBO) on platelet physiology. DESIGN AND METHODS: Human platelets were exposed to HBO (97.7% O(2), balance CO(2) at 2.2 ata) or control (CON; 5% CO(2), balance air at 1 ata) for 90 min, and analyzed for aggregation, protein release, ()NO production, and activation. RESULTS: HBO induced 29.8+/-3.0% of platelets to aggregate compared with CON (5.5+/-0.9%). Proteins observed to be released in greater abundance from HBO- compared with CON-treated platelets included 14-3-3 zeta and alpha-2-macroglobulin. Release of ()NO by platelets was unaffected following exposure to HBO, as was platelet activation as measured by surface expression of PECAM-1, CD62P and the activated form of alpha(IIB)beta(IIIa). CONCLUSIONS: Exposure to HBO induces both platelet aggregation and protein release. Further study will better define the precise mechanisms and effects of HBO on platelet activation.

Temporal transcription of two antennapedia class homeobox genes in the marine copepod Calanus helgolandicus.

DNA sequence information has been obtained for 2 genes implicated in playing critical roles in copepod development. The first ( Cal-antp) was obtained by screening a Calanus helgolandicus genomic library. It shows homology to Antennapedia homeobox genes from a number of organisms including the centipede (76.8% over 250 bp) and the brine shrimp (75.5% over 220 bp). A second sequence (Hox 12) was obtained through reverse transcription polymerase chain reaction (PCR) of a solid-phase cDNA library constructed from Calanus eggs, and subsequent inverse PCR using a genomic DNA template. The Hox 12 sequence is homologous to the Antennapedia class of genes and shares homology with the chicken HoxB3 gene (71% over 187 bp) and with the fruit fly Sex Combs Reduced ( Scr) homeotic gene (66% identity over 223 bp). Using sequence data from Cal-Antp and Hox 12, specific primers were designed to investigate the temporal expression of these genes by PCR analysis of 10 stage-specific cDNA libraries.