Dexmedetomidine enhances ropivacaine-induced sciatic nerve injury in diabetic rats.

BACKGROUND: Previous studies suggest that dexmedetomidine has a protective effect against local anaesthetic-induced nerve injury in regional nerve blocks. Whether this potentially protective effect exists in the context of diabetes mellitus is unknown. METHODS: A diabetic state was established in adult male Sprague-Dawley rats with intraperitoneal injection of streptozotocin. Injections of ropivacaine 0.5%, dexmedetomidine 20 µg kg-1 (alone and in combination), or normal saline (all in 0.2 ml) were made around the sciatic nerve in control and diabetic rats (n=8 per group). The duration of sensory and motor nerve block and the motor nerve conduction velocity (MNCV) were determined. Sciatic nerves were harvested at post-injection day 7 and assessed with light and electron microscopy or used for pro-inflammatory cytokine measurements. RESULTS: Ropivacaine and dexmedetomidine alone or in combination did not produce nerve fibre damage in control non-diabetic rats. In diabetic rats, ropivacaine induced significant nerve fibre damage, which was enhanced by dexmedetomidine. This manifested with slowed MNCV, decreased axon density, and decreased ratio of inner to outer diameter of the myelin sheath (G ratio). Demyelination, axon disappearance, and empty vacuoles were also found using electron microscopy. An associated increase in nerve interleukin-1ß and tumour necrosis factor-α was also seen. CONCLUSIONS: Ropivacaine 0.5% causes significant sciatic nerve injury in diabetic rats that is greatly potentiated by high-dose dexmedetomidine. Although the dose of dexmedetomidine used in this study is considerably higher than that used in clinical practice, our data suggest that further studies to assess ropivacaine (alone and in combination with dexmedetomidine) use for peripheral nerve blockade in diabetic patients are warranted.

Characterization of friction and moisture of porcine lingual tissue in vitro in response to artificial saliva and mouthwash solutions.

BACKGROUND: The purpose of this research is to characterize the effects of mouthwash solutions on oral friction and moisture using a quantitative in vitro approach. MATERIALS AND METHODS: The frictional coefficient of in vitro porcine tongue samples was measured using a magnetic levitation haptic device equipped with a custom tactor designed to mimic human skin. A commercially available moisture meter was used to measure moisture content of the samples. Tongue samples were first tested before treatment, then after application of saliva (either human or artificial), and again after application of 1 of 11 different mouthwash solutions. RESULTS: The data indicate that the samples treated with artificial saliva vs real saliva have comparable friction coefficient and moisture content. Furthermore, the moisture and friction coefficient remain relatively constant for up to 60 minutes after exposure to ambient conditions. Samples treated with artificial saliva have an average friction coefficient in the range of 0.70-0.80. Application of mouthwash solutions produced an average friction coefficient of 0.39-0.49 but retained the high moisture content of the artificial salivary layer. Several mouthwash solutions resulted in statistically significant differences in the friction coefficient relative to each other. CONCLUSION: The results of this study demonstrate that a magnetic levitation device can be an effective tool for in vitro oral tribology and that artificial saliva is an effective substitute for real saliva in extended in vitro experiments. The application of mouthwash generally reduces the coefficient of friction of the tongue samples while preserving a relatively high moisture level, and some mouthwashes reduce friction significantly more than others.

Overcoming the dichotomy of implementing societal flood risk management while conserving instream fish habitat - A long-term study from a highly modified urban river.

Flood Risk Management (FRM) is often essential to reduce the risk of flooding to properties and infrastructure in urban landscapes, but typically degrades the habitats required by many aquatic animals for foraging, refuge and reproduction. This conflict between flood risk management and biodiversity is driven by conflicting directives, such as the EU Floods and Water Framework Directives, and has led to a requirement for synergistic solutions for FRM that integrate river restoration actions. Unfortunately, ecological monitoring and appraisal of combined FRM and river restoration works is inadequate. This paper uses a case study from the River Don in Northern England to evaluate the effects of the FRM and subsequent river restoration works on instream habitat and the associated fish assemblage over an 8-year period. Flood risk management created a homogeneous channel but did not negatively affect fish species composition or densities, specifically brown trout. Densities of adult brown trout were comparable pre and post-FRM, while densities of juvenile bullhead and brown trout increased dramatically post FRM. River restoration works created a heterogeneous channel but did not significantly improve species composition or brown trout density. Species composition post-river restoration works returned to that similar to pre-FRM over a short-term period, but with improved numbers of juvenile bullhead. Although habitat complexity increased after river restoration works, long-term changes in species composition and densities were marginal, probably because the river reset habitat complexity within the time framework of the study.

Haptic characterization of human skin in vivo in response to shower gels using a magnetic levitation device.

BACKGROUND/PURPOSE: Skin products such as shower gels have a direct impact on skin health and wellness. Although qualitative haptic characterization through explicit, verbal measures in consumer studies are often sufficient for general comparison on consumer perceived skin feel, a quantitative approach is desired to characterize minute changes in skin condition in response to various skin products. Prior research has sought to characterize the haptic properties of human skin in vitro and in vivo, but very few studies have compared the haptic effects of commercial skin products having relatively similar formulations. In addition, related studies have typically utilized simple, low-precision devices and fixtures. The purpose of this study was to use a precision magnetic levitation haptic device to characterize the frictional properties of human skin in vivo before, during, and after treatment with commercially available shower gels, to capture the entire cycle of consumer experience on skin feel. METHODS: A hybrid force-position control algorithm was used to control a precision magnetic levitation haptic device with silicone tactor to stroke the human skin (on the volar forearm) in vivo. Position and force data were collected from 32 human subjects using eight different commercially available shower gels, while stroking the skin before, during, and after treatment. The data were analyzed to produce coefficients of friction and viscous damping constant, which were used as metrics for comparing the effects of each shower gel type. Other factors investigated include skin test location, order, and subject age and gender. RESULTS: Results showed significant differences between the effects of eight various shower gels, especially after accounting for variance between subjects. Most notably, Shower Gel four with high level of petrolatum, along with Shower Gels five and six with low levels of castoryl maleate (a skin lipid analog), as well as Shower Gel two with high levels of vegetable oils yielded higher skin coefficients of friction 20 min after treatment, indicating higher levels of skin hydration than other shower gels without either high levels of skin beneficial agents or low levels of castoryl maleate. Conversely, Shower Gel eight treatment yielded the lowest skin coefficient of friction both immediately after rinsing and 20 min after treatment. In addition, when applied to the skin as un-lathered gels, Shower Gels six and seven with acrylate polymers yielded viscous damping constants twice that of other gels, while Shower Gel three yielded the lowest. When lathered into foam on skin, Shower Gel eight yielded the highest viscous damping constant, while Shower Gel three, along with Shower Gels one and five yielded lower values than others. CONCLUSION: The results of this study show that different shower gels do have significant measurable differences in their effects on skin properties, and that using a high-precision haptic device can be a useful tool for quantifying the haptic properties of skin in vivo.

Guidelines for the use of cell lines in biomedical research.

Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise.

Perceived barriers and facilitators to physical activity in men with prostate cancer: possible influence of androgen deprivation therapy.

While physical activity is beneficial for men with prostate cancer, too few perform sufficient activity for such benefit. This study examined perceptions of men with prostate cancer of their barriers and facilitators to physical activity, and how androgen deprivation therapy (ADT) may influence these perceptions. Two focus groups were conducted, involving six ADT and eight non-ADT patients respectively. Data were transcribed verbatim and themes developed using a general inductive thematic approach. Facilitators to physical activity common to both groups of cancer survivors included clinician and spousal involvement, with pre-existing co-morbidities and increased age cited as barriers by both groups. The ADT subgroup cited personal involvement as a facilitator to physical activity, with fatigue, reduced motivation and a relative lack of specific advice from their clinician as additional barriers. The non-ADT subgroup had no additional facilitators to physical activity but cited time constraints as a barrier. These results highlight the important role that cancer clinicians and spouses play in promoting physical activity for men with prostate cancer and how ADT may influence their other facilitators and barriers. As physical activity is beneficial for prostate cancer survivors, especially those on ADT, cancer clinicians should regularly discuss physical activity with their patients.

Seeing the wood through the trees: the current state of higher systematics in the Strepsirhini.

Strepsirhines comprise 10 living or recently extinct families, ≥50% of extant primate families. Their phylogenetic relationships have been intensively studied, but common topologies have only recently emerged; e.g. all recent reconstructions link the Lepilemuridae and Cheirogaleidae. The position of the indriids, however, remains uncertain, and molecular studies have placed them as the sister to every clade except Daubentonia, the preferred sister group of morphologists. The node subtending Afro-Asian lorisids has been similarly elusive. We probed these phylogenetic inconsistencies using a test data set including 20 strepsirhine taxa and 2 outgroups represented by 3,543 mtDNA base pairs, and 43 selected morphological characters, subjecting the data to maximum parsimony, maximum likelihood and Bayesian inference analyses, and reconstructing topology and node ages jointly from the molecular data using relaxed molecular clock analyses. Our permutations yielded compatible but not identical evolutionary histories, and currently popular techniques seem unable to deal adequately with morphological data. We investigated the influence of morphological characters on tree topologies, and examined the effect of taxon sampling in two experiments: (1) we removed the molecular data only for 5 endangered Malagasy taxa to simulate 'extinction leaving a fossil record'; (2) we removed both the sequence and morphological data for these taxa. Topologies were affected more by the inclusion of morphological data only, indicating that palaeontological studies that involve inserting a partial morphological data set into a combined data matrix of extant species should be interpreted with caution. The gap of approximately 10 million years between the daubentoniid divergence and those of the other Malagasy families deserves more study. The apparently contemporaneous divergence of African and non-daubentoniid Malagasy families 40-30 million years ago may be related to regional plume-induced uplift followed by a global period of cooling and drying.

A role for class 3 semaphorins in prostate cancer.

BACKGROUND: Class 3 semaphorins are secreted proteins that act as guidance cues for migrating cells via their transmembrane receptors plexins and neuropilins. Semaphorins have a role in cancer affecting tumor progression both directly, and indirectly by affecting angiogenesis. METHODS: The expression of semaphorins and their receptors in prostate cancer cell lines and tissue was determined by RT-PCR, Western blotting and immunohistochemistry. The effect of Sema3E on prostate cancer cell lines was determined by adhesion assays and transwell migration assays. RESULTS: Semaphorins and their receptors, plexins and neuropilins, are widely co-expressed in prostate cancer cell lines and tissue with a significant overexpression of Sema3E in tumor tissue. Sema3E affected integrin-mediated adhesion to fibronectin of prostate cancer cells, and inhibited their motility. Expression of Sema3C was upregulated and Sema3A and Sema3E were down regulated in prostate cells by hypoxia, consistent with an additional role for Sema3A and 3E as anti-angiogenic factors in prostate cancer. CONCLUSIONS: Semaphorin 3E is aberrantly expressed in prostate cancer and affects adhesion and motility of prostate cancer cells, indicating a role for the Sema3E/PlexinD1 signaling pathway in prostate cancer and identifying a new possible target for therapy.

Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys.

Apoptosis is a morphologically defined form of programmed cell death seen in a variety of circumstances, including immune cell selection, carcinogenesis and development. Apoptosis has very recently been seen after ischemic or traumatic injury to the central nervous system (CNS), suggesting that active cell death as well as passive necrosis may mediate damage after CNS injury. After spinal cord injury (SCI) in the rat, typical post-traumatic necrosis occurred, but in addition, apoptotic cells were found from 6 hours to 3 weeks after injury, especially in the spinal white matter. Apoptotic cells were positive for oligodendrocyte markers. After SCI in monkeys, apoptotic cells were found within remote degenerating fiber tracts. Both secondary degeneration at the site of SCI and the chronic demyelination of tracts away from the injury appear to be due in part to apoptosis. As cytokines have been shown to mediate oligodendrocyte death in vitro, it seems likely that chronic demyelination after CNS injury shares features with chronic degenerative disorders like multiple sclerosis.

The gunshot-related injuries in trauma (GRIT) study: A profile of patients affected by gunshot-related orthopaedic injuries across South Africa.

BACKGROUND: South Africa (SA) has one of the highest gun-related mortality rates in the world - 20 people per day. The available data, however, do not reflect the substantial number of patients suffering non-lethal firearm injuries. Gunshot-related injury has been recognised as a highly costly healthcare problem by individual treating centres in SA and other countries; however, no 'national picture' has been examined in detail. OBJECTIVES: To explore the burden of gunshot-related orthopaedic injuries across SA. METHODS: A multicentre research network was established in SA, and 37 orthopaedic units across 9 provinces participated. A prospective, observational cohort study was performed during a 2-week period in 2019. Patients were screened, enrolled and reported by local orthopaedic teams. Patients were included if they had at least one acute gunshot-related orthopaedic fracture referred to the orthopaedic service. Patients were asked additional questions around baseline health-related quality-of-life (HRQOL) and personal circumstances. Follow-up was at 8 weeks after injury. RESULTS: Thirty-seven centres enrolled 135 patients over the 2-week study period. Western Cape Province had the highest number of reported cases (n=52; 39%), followed by Gauteng (n=35; 26%) and KwaZulu-Natal (n=29; 21%). The median age of patients was 30.5 years and the majority were male (89%). Forty-three percent of patients had been either shot or stabbed prior to this injury. Fifty-two percent of all patients required fracture fixation surgery and 11% required wound debridement without fracture fixation. HRQOL data were collected successfully at baseline, but follow-up data were available for <25% of cases. CONCLUSIONS: Gunshot-related orthopaedic injuries represent a significant burden of disease in the SA healthcare environment. This study highlights several areas for further research in the management of the injuries and associated outcomes.

Vertically acquired HIV diagnosed in adolescence and early adulthood in the United Kingdom and Ireland: findings from national surveillance.

OBJECTIVE: The aim of the study was to describe the characteristics of young people with vertically acquired HIV diagnosed aged > or =13 years. METHODS: A retrospective review of HIV diagnoses reported to well-established national paediatric and adult HIV surveillance systems in the United Kingdom/Ireland was conducted. RESULTS: Forty-two young people with vertically acquired HIV diagnosed aged > or =13 years were identified; 23 (55%) were female, 40 (95%) were black African and 36 (86%) were born in sub-Saharan Africa. The median age at HIV diagnosis was 14 years (range, 13-20 years). Half of the patients presented with symptoms; the remainder were screened for HIV following diagnosis of a relative. The median CD4 count at diagnosis was 210 cells/microL (range, 0-689 cells/microL), 12 patients (29%) were diagnosed with AIDS at HIV diagnosis or subsequently, and 34 (81%) started combination antiretroviral therapy (ART), most (31 of 34) within a year of diagnosis. CONCLUSION: A small number of young people with vertically acquired HIV survive childhood without ART and are diagnosed at age > or =13 years in the United Kingdom/Ireland. Half of the patients were asymptomatic, highlighting the importance of considering HIV testing for all offspring of HIV-infected women, regardless of age or symptoms. Increased awareness among clinicians and parents is required to reduce delayed presentation with advanced disease and to avoid onward transmission as these young people become sexually active.

Use of chemokine receptors by poxviruses.

Chemokine receptors serve as portals of entry for certain intracellular pathogens, most notably human immunodeficiency virus (HIV). Myxoma virus is a member of the poxvirus family that induces a lethal systemic disease in rabbits, but no poxvirus receptor has ever been defined. Rodent fibroblasts (3T3) that cannot be infected with myxoma virus could be made fully permissive for myxoma virus infection by expression of any one of several human chemokine receptors, including CCR1, CCR5, and CXCR4. Conversely, infection of 3T3-CCR5 cells can be inhibited by RANTES, anti-CCR5 polyclonal antibody, or herbimycin A but not by monoclonal antibodies that block HIV-1 infection or by pertussis toxin. These findings suggest that poxviruses, like HIV, are able to use chemokine receptors to infect specific cell subtypes, notably migratory leukocytes, but that their mechanisms of receptor interactions are distinct.

Cost-effectiveness of negative-pressure wound therapy in adults with severe open fractures of the lower limb: evidence from the WOLLF randomized controlled trial.

AIMS: The aim of this study was to estimate the cost-effectiveness of negative-pressure wound therapy (NPWT) in comparison with standard wound management after initial surgical wound debridement in adults with severe open fractures of the lower limb. PATIENTS AND METHODS: An economic evaluation was conducted from the perspective of the United Kingdom NHS and Personal Social Services, based on evidence from the 460 participants in the Wound Management of Open Lower Limb Fractures (WOLLF) trial. Economic outcomes were collected prospectively over the 12-month follow-up period using trial case report forms and participant-completed questionnaires. Bivariate regression of costs (given in £, 2014 to 2015 prices) and quality-adjusted life-years (QALYs), with multiple imputation of missing data, was conducted to estimate the incremental cost per QALY gained associated with NPWT dressings. Sensitivity and subgroup analyses were undertaken to assess the impacts of uncertainty and heterogeneity, respectively, surrounding aspects of the economic evaluation. RESULTS: The base case analysis produced an incremental cost-effectiveness ratio of £267 910 per QALY gained, reflecting higher costs on average (£678; 95% confidence interval (CI) -£1082 to £2438) and only marginally higher QALYS (0.002; 95% CI -0.054 to 0.059) in the NPWT group. The probability that NPWT is cost-effective in this patient population did not exceed 27% regardless of the value of the cost-effectiveness threshold. This result remained robust to several sensitivity and subgroup analyses. CONCLUSION: This trial-based economic evaluation suggests that NPWT is unlikely to be a cost-effective strategy for improving outcomes in adult patients with severe open fractures of the lower limb. Cite this article: Bone Joint J 2019;101-B:1392-1401.

Validation of a prospective cohort study of older adults with hip fractures.

AIMS: This study sought to determine the proportion of older adults with hip fractures captured by a multicentre prospective cohort, the World Hip Trauma Evaluation (WHiTE), whether there was evidence of selection bias during WHiTE recruitment, and the extent to which the WHiTE cohort is representative of the broader population of older adults with hip fractures. PATIENTS AND METHODS: The characteristics of patients recruited into the WHiTE cohort study were compared with those treated at WHiTE hospitals during the same timeframe and submitted to the National Hip Fracture Database (NHFD). RESULTS: Patients recruited to WHiTE were more likely to be admitted from their own home (83.5% vs 80.2%; p < 0.001) and to have a higher median Abbreviated Mental Test Score (AMTS) (9 (interquartile range (IQR) 6 to 10) vs 9 (IQR 5 to 10); p < 0.001) than those who were not recruited. In terms of WHiTE cohort generalizability, participating hospitals included a greater proportion of Major Trauma Centres (47.8% vs 7.8%) and large hospitals (997 (IQR 873 to 1290) vs 707 (459 to 903) beds) with high-volume Emergency Departments (median annual attendances of 43 981 (IQR 37 147 to 54 385) vs 35 964 (IQR 26 229 to 50 551)). However, there were few differences in baseline characteristics between patients in the WHiTE cohort and those recorded in the NHFD. CONCLUSION: There is evidence of a weak selection bias towards recruiting fitter patients within the WHiTE cohort, which will help to put into context the findings of future studies. We conclude that the patients within the WHiTE cohort are representative of the national population of older adults with hip fractures throughout England, Wales, and Northern Ireland. Cite this article: Bone Joint J 2019;101-B:708-714.

Hypomethylation of WNT5A, CRIP1 and S100P in prostate cancer.

Oligoarray analysis of a matched pair of prostate cancer and normal cell lines derived from the same radical prostatectomy specimen identified 113 candidate hypomethylated genes that were overexpressed in the cancer cells and contained CpG islands. Hypomethylation of wingless-related MMTV integration site 5A (WNT5A), S100 calcium-binding protein P (S100P) and cysteine-rich protein 1(CRIP1) was confirmed in the cancer cells by bisulfite sequencing. Treatment of the corresponding normal prostate epithelial cells 1542-NPTX with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-aza-CdR) induced higher levels of mRNA expression and partial loss of methylation on these genes. Primary prostate cancers were tested using methylation-specific polymerase chain reaction. WNT5A was hypomethylated in 11/17 (65%) tumors, S100P in 8/16 (50%) and CRIP1 in 13/20 (65%). Bisulfite sequencing of a section of the 5' untranslated region (UTR) of WNT5A revealed that three CpG sites (15, 24 and 35) were consistently methylated (93%) in the normal cell line and normal tissues, but not in the prostate cancer cell line and eight primary prostate cancers. Multiple putative binding sites for the transcription factors SP1 and AP-2 were found adjacent to CpG sites 15 and 24. A putative c-Myb binding site was located within the CpG site 35. Anti-c-Myb antibody co-precipitation with WNT5A was methylation-sensitive in 1542-NPTX cells. It is likely that an epigenetic mechanism regulates WNT5A expression in prostate cancer.

Dynamics of gene expression for a hippocampal glycoprotein elevated in Alzheimer's disease and in response to experimental lesions in rat.

A hippocampal poly(A) RNA, pADHC-9, was cloned by differential screening of a human hippocampal cDNA library. By RNA blot analysis, pADHC-9 was elevated 2-fold in Alzheimer's disease hippocampus. In situ analyses identified pADHC-9 expression in pyramidal and non-pyramidal cells of the hippocampus and entorhinal cortex. Nucleotide sequence analysis identified pADHC-9 as a potential human homolog of rat sulfated glycoprotein 2 (SGP-2). SGP-2 expression increased in rat hippocampus following experimental lesions that mimic intrinsic neuronal loss and/or deafferentation. The function of pADHC-9 in brain has not been defined, but in serum, a similar protein inhibits complement-dependent cytolysis. Increased expression of pADHC-9 in Alzheimer's disease hippocampus may be a compensatory response mounted to retard a complement-driven neurodegenerative cascade.

Economic outcomes associated with deep surgical site infection in patients with an open fracture of the lower limb.

AIMS: The aim of this study was to estimate economic outcomes associated with deep surgical site infection (SSI) in patients with an open fracture of the lower limb. PATIENTS AND METHODS: A total of 460 patients were recruited from 24 specialist trauma hospitals in the United Kingdom Major Trauma Network. Preference-based health-related quality-of-life outcomes, assessed using the EuroQol EQ-5D-3L and the 6-Item Short-Form Health Survey questionnaire (SF-6D), and economic costs (£, 2014/2015 prices) were measured using participant-completed questionnaires over the 12 months following injury. Descriptive statistics and multivariate regression analysis were used to explore the relationship between deep SSI and health utility scores, quality-adjusted life-years (QALYs), and health and personal social service (PSS) costs. RESULTS: Deep SSI was associated with lower EQ-5D-3L derived QALYs (adjusted mean difference -0.102, 95% confidence interval (CI) -0.202 to 0.001, p = 0.047) and increased health and social care costs (adjusted mean difference £1950; 95% CI £1383 to £5285, p = 0.250) versus patients without deep SSI over the 12 months following injury. CONCLUSION: Deep SSI may lead to significantly impaired health-related quality of life and increased economic costs. Our economic estimates can be used to inform clinical and budgetary service planning and can act as reference data for future economic evaluations of preventive or treatment interventions. Cite this article: Bone Joint J 2018;100-B:1506-10.

Defective repair of cisplatin-induced DNA damage caused by reduced XPA protein in testicular germ cell tumours.

Metastatic cancer in adults usually has a fatal outcome. In contrast, advanced testicular germ cell tumours are cured in over 80% of patients using cisplatin-based combination chemotherapy [1]. An understanding of why these cells are sensitive to chemotherapeutic drugs is likely to have implications for the treatment of other types of cancer. Earlier measurements indicate that testis tumour cells are hypersensitive to cisplatin and have a low capacity to remove cisplatin-induced DNA damage from the genome [2] [3]. We have investigated the nucleotide excision repair (NER) capacity of extracts from the well-defined 833K and GCT27 human testis tumour cell lines. Both had a reduced ability to carry out the incision steps of NER in comparison with extracts from known repair-proficient cells. Immunoblotting revealed that the testis tumour cells had normal amounts of most NER proteins, but low levels of the xeroderma pigmentosum group A protein (XPA) and the ERCC1-XPF endonuclease complex. Addition of XPA specifically conferred full NER capacity on the testis tumour extracts. These results show that a low XPA level in the testis tumour cell lines is sufficient to explain their poor ability to remove cisplatin adducts from DNA and might be a major reason for the high cisplatin sensitivity of testis tumours. Targeted inhibition of XPA could sensitise other types of cells and tumours to cisplatin and broaden the usefulness of this chemotherapeutic agent.

Discrete human dihydrofolate reductase gene transcripts present in polysomal RNA map with their 5' ends several hundred nucleotides upstream of the main mRNA start site.

The 5' ends of dihydrofolate reductase (DHFR)-specific transcripts have been mapped in the 5'-flanking region of the amplified DHFR gene of the human methotrexate-resistant cell line 6A3 by primer extension and S1 protection experiments. The main 5' end, at position -71 relative to the first nucleotide of the DHFR reading frame, corresponds to the recently identified main transcription initiation site for the DHFR gene and pertains to transcripts representing approximately 99% of the DHFR-specific polysomal polyadenylic acid-containing RNA, and including the previously described DHFR mRNAs with sizes of 3.8, 1.0, and 0.8 kilobases. At least six other minor 5' ends have been mapped to nucleotide positions -449 to -480 upstream of the DHFR gene and pertain to approximately 1% of the DHFR-specific polysomal polyadenylic acid-containing RNA. These upstream initiating transcripts appear to include five major discrete species with sizes of 4.3, 3.8, 3.1, 2.1, and 1.0 kilobases and four minor ones with sizes of 7.3, 5.0, 1.4, and 0.8 kilobases. These species, with the exception of those of 3.1- and 2.1-kilobase sizes, also have been found in VA2-B cells, the parental line of 6A3, and in HeLa cells. The upstream initiating transcripts present in all three cell lines are increased in amount in 6A3 cells as compared with the other cell lines, in about the same proportion as the three identified DHFR mRNAs.