Influence of multi-lumen extensions on fluid flow through intravenous cannulae.

Multi-lumen extensions used to infuse multiple fluids via a single intravenous cannula might increase resistance and so limit the flow that can be achieved. We constructed low-pressure and high-pressure models and compared the effect of two different multi-lumen extensions on flow rate. Both multi-lumen extensions reduced flows by up to 76% (p < 0.001). The effect was greatest with large cannulae and in the high-pressure model, with the longer and narrower extension most impeding flow. Multi-lumen extensions can therefore significantly impede fluid flow, and should be avoided or removed when rapid infusion is required. These effects are less important in paediatric anaesthesia where smaller cannulae are used. Manufacturers should include internal diameter or flow effects on the packaging of these extensions to assist clinicians in making such decisions.

NBT-PABA test to assess efficiency and kinetics of substituted proteolytic enzyme action in pancreatic duct ligated minipigs.

The NBT-PABA test is an established method for diagnosis of pancreatic exocrine insufficiency. In the present study the NBT-PABA test was used to test and compare the efficacy of two multienzyme preparations (product A and B) differing in galenic preparation in minipigs in which pancreatic exocrine insufficiency (PEI) was induced by pancreatic duct ligation. Without enzyme substitution no distinct increase in PABA was found in blood after oral administration of NBT-PABA. Administration of both enzyme preparations led to a clear dose dependent rise in PABA-concentrations in blood. Interestingly, the two preparations showed different time curves of serum PABA concentration, indicating differences in the kinetic of proteolytic enzyme action. It is concluded that the NBT-PABA test can be a very useful test for indirectly evaluating proteolytic enzyme efficacy in vivo, and also gives information about the kinetics of enzyme action, not only the end-result of enzyme action (like digestibility trials which were used traditionally). A single test is performed in a few hours and there is no need for fistulated animals.

Functional identification of secondary mutations inducing autonomous growth in synergy with a truncated interleukin-3 receptor: implications for multi-step oncogenesis.

OBJECTIVE: A truncated common beta chain (Deltabeta(C)) of the interleukin-3 (IL-3) receptor complex was previously identified as a key factor in inducing autonomous growth of IL-3-independent mutants. Expression of Deltabeta(C) in IL-3-dependent hematopoietic cells does not result in immediate factor-independent growth, but increases the frequency of obtaining autonomous mutants by three to four orders of magnitude. This study was designed to delineate the mechanisms by which Deltabeta(C) increases the frequency to autonomous growth. DESIGN AND METHODS: Retroviral vectors were used to express Deltabeta(C) into IL-3-dependent myeloid cells, which were then tested for factor-independent growth. To determine if secondary genetic events were required for conversion to autonomous growth, elements of the Cre-loxP recombinant system were used to excise Deltabeta(C) in factor-independent clones. RESULTS: Excision of Deltabeta(C) in factor-independent clones revealed two types of phenotypes: reversion to factor-dependent growth (1/8) or continued IL-3-dependent growth (7/8). Analysis of cells that remained factor independent revealed constitutive activation of STAT5, not observed in factor-dependent revertants. Analysis of revertant cells demonstrated the presence of interacting secondary mutations that synergize with Deltabeta(C)-induced proliferation. A cysteine residue within the truncated extracellular domain of Deltabeta(C) was also found to be required for its oncogenic potential, supporting a model of dimerization for receptor activation. CONCLUSIONS: The high incidence of obtaining factor-independent mutants from cells expressing Deltabeta(C) results from the selection of mutations that either complement Deltabeta(C) expression to promote proliferation or that singly or in synergy with other secondary mutations negate the requirement of Deltabeta(C) expression for proliferation.

In human keratinocytes the Common Deletion reflects donor variabilities rather than chronologic aging and can be induced by ultraviolet A irradiation.

Mitochondrial DNA mutations play a major role in human aging processes and degenerative diseases. The most frequently reported marker for mutations of the mitochondrial DNA in human skin is a 4977 bp large-scale deletion, called the Common Deletion. Although this deletion is rarely detectable and constitutes only one example of the multitude of about 50,000 known mutations in mitochondrial DNA, it can represent "the tip of the iceberg" of all types of mitochondrial DNA mutations. We established a quantitative real-time polymerase chain reaction assay to detect the Common Deletion in vitro as well as in vivo/ex vivo. In contrast to previous studies, we were able to demonstrate that the Common Deletion is frequently abundant in keratinocytes isolated from various donors. Quantitative analysis of the mutation indicated interperson variations but obviously no relation to the donors' ages. Prolonged proliferation of keratinocytes led to a distinct reduction in the amount of the Common Deletion. Single ultraviolet A irradiation (12 J per cm2 and 15 J per cm2) neither in vitro nor in vivo increased the incidence of the mutation in keratinocytes, whereas repetitive irradiation resulted in a clear increase in vitro. Again, prolonged cultivation of these irradiated cells caused a significant reduction in the amounts of the deletion. In view of these results, the Common Deletion appears to be a useful marker rather for ultraviolet-A-induced alterations than for chronologic aging in human skin keratinocytes.

Two different macronuclear EF-1 alpha-encoding genes of the ciliate Euplotes crassus are very dissimilar in their sequences, copy numbers and transcriptional activities.

Genes (EFA) encoding the translation elongation factor EF-1 alpha (EFA) or the prokaryotic homolog EFTu frequently occur in multiple copies in the same organism. This has been interpreted either in terms of a potential of differential gene expression during different phases of development, or as gene dosage adaptation to the need of high-level production of the gene products. Since ciliates can differentially amplify their genes, the latter argument would lead to the expectation of only one EFA gene in the macronucleus. However, we have found two such genes which strongly differ in both copy number and codon usage. Both transcripts are detectable at very different levels. The expression of the genes takes place both in the vegetative and sexual phases, i.e.,during conjugation.

Semiquantitative chemiluminescent detection of UV-B-induced point mutations in the p53 tumor-suppressor gene.

The technique of allele-specific PCR (AS-PCR) enables the detection of a small number of mutant alleles in a large number of wild-type (WT) alleles. We used the AS-PCR technique and Southern blotting, using a nonradioactive labeled probe to analyze the formation of point mutations in the tumor-suppressor gene p53 of primary keratinocytes after UV-B irradiation. These permanent mutations resulting from CC dimers occur at distinct "hot-spots", one of which is affected in the human keratinocyte cell line HaCaT. This enabled us to establish the method with a defined positive control template, which also allowed semiquantitative determination of the mutation frequency. This, and the determination of the detection limit, was done with the use of serial dilutions of WT genomic DNA from primary keratinocytes with mutant genomic HaCaT DNA in the AS-PCR assay.

Induction of mRNA for matrix metalloproteinase 1 and tissue inhibitor of metalloproteinases 1 in human skin in vivo by solar simulated radiation.

Repeated exposure to solar ultraviolet radiation results in premature skin aging due, in part, to the degradation of dermal collagen by fibroblast collagenase (matrix metalloproteinase 1 [MMP-1]). We have established TaqMan reverse transcription (RT) polymerase chain reaction (PCR) systems to quantify the messenger RNA (mRNA) expression of MMP-1 and its specific inhibitor TIMP-1 in human buttock skin exposed in vivo to solar simulated radiation (SSR). A time-course study (n = 6) with two minimal erythema doses (MED) of SSR showed maximal induction of MMP-1 and TIMP-1 at 24 h. A dose-response study (n = 6) sampled at 24 h revealed that doses of about 1 MED were necessary to induce expression of MMP-1 mRNA, and our data suggest that the response is saturated at about 2 MED. We also investigated SSR-induced gene expression in the dermis and epidermis separately (n = 5). MMP-1 was present in both tissues, but TIMP-1 was only detected in the dermis. In general, we could only measure MMP-1 mRNA in the nonirradiated control skin of volunteers who were smokers. We hypothesize very large interpersonal variation with MMP-1 induction compared with TIMP-1 which was detected in all the control sites. This suggests a lack of relationship between MMP-1 and TIMP-1 mRNA expression. The large donor variability for MMP-1 in all the studies demonstrates that it is important to analyze gene expression individually.

P53-dependent UVB responsiveness of human keratinocytes can be altered by cultivation on cell cycle-arrested dermal fibroblasts.

In cultured human keratinocytes, the tumor suppressor p53 acts as a control element in the protective response to UVB radiation and is affected by a variety of factors linked to cellular adhesion and differentiation. Because keratinocytes within the epidermis are not a homogeneous population but differ in their proliferative capacity and differentiation status, we compared the UVB responsiveness of primary keratinocyte populations isolated from various skin biopsies using p53 expression as a marker for their sensitivity to UVB. Besides keratinocytes exhibiting a UVB dose- and time-dependent upregulation of p53, keratinocyte populations were detected with high p53 expression levels even without irradiation. Such keratinocytes did not regulate p53 expression in response to UVB. Furthermore their p53-mediated UVB response was influenced by cocultivation with human dermal fibroblasts (HDF) but not with cell cycle-arrested human normal keratinocytes or HaCaT keratinocytes. When these cells were cultivated together with arrested HDF, they did not only reveal increased p53 expression levels after UVB treatment but also a more pronounced transcriptional activation of the p53 downstream target gene p21. These findings indicate that the UVB response of keratinocytes, specifically the activation of the tumor suppressor p53, is heterogeneous and can be affected by growth conditions.

Analysis of UV-B-induced DNA damage and its repair in heat-shocked skin cells.

The heat-shock response is a cellular defence mechanism against environmental stresses that is evolutionarily conserved from bacteria to man. Numerous reports demonstrate the beneficial effects of heat-shock protein induction on cell survival under toxic or oxidative stress, e.g., in cardiac and cerebral ischemia or prior to organ transplantation. However, there is little data on the effects of heat treatment on damage caused by UV irradiation. Applying three independent techniques, we have tested the influence of thermal pretreatment of skin cells (1 h, 43 degrees C) on the initial extent of UV-B-induced DNA damage and its subsequent repair. For cultured human epidermal keratinocytes and dermal fibroblasts we can show reduced levels of nucleotide-excision-repair-associated DNA strand incision in the comet assay. Moreover, immunostaining and flow cytometric quantitation of thymidine dimers immediately and one day after irradiation, respectively, reveal that the initial DNA damage is not (keratinocytes) or only moderately (fibroblasts) lower in heat-shocked cells as compared to untreated controls. However, excision repair of dimers is significantly attenuated during the first 24 h in both cell types. Furthermore, using a modified host-cell reactivation assay, we are able to demonstrate that repair of UV-B-damaged plasmid DNA is lower if the transfected cells are previously heat shocked. In summary, heat treatment (1 h, 43 degrees C) inducing heat-shock proteins reduces nucleotide excision repair of UV-B-mediated DNA lesions in fibroblasts and keratinocytes during the following 24 h. This is not necessarily caused by elevated heat-shock protein levels themselves. Possibly the direct thermal damage of repair enzymes is more severe than the potential protective effects of heat-shock proteins.

Coenzyme Q10, a cutaneous antioxidant and energizer.

The processes of aging and photoaging are associated with an increase in cellular oxidation. This may be in part due to a decline in the levels of the endogenous cellular antioxidant coenzyme Q10 (ubiquinone, CoQ10). Therefore, we have investigated whether topical application of CoQ10 has the beneficial effect of preventing photoaging. We were able to demonstrate that CoQ10 penetrated into the viable layers of the epidermis and reduce the level of oxidation measured by weak photon emission. Furthermore, a reduction in wrinkle depth following CoQ10 application was also shown. CoQ10 was determined to be effective against UVA mediated oxidative stress in human keratinocytes in terms of thiol depletion, activation of specific phosphotyrosine kinases and prevention of oxidative DNA damage. CoQ10 was also able to significantly suppress the expression of collagenase in human dermal fibroblasts following UVA irradiation. These results indicate that CoQ10 has the efficacy to prevent many of the detrimental effects of photoaging.

Omental herniation through a 3-mm umbilical trocar site: unmasking a hidden umbilical hernia.

BACKGROUND: The incidence of umbilical hernia following laparoscopic surgery varies from 0.02-3.6%. The incidence of pre-existing fascial defects, however, may be as high as 18% in patients undergoing abdominal laparoscopic surgery. Previous recommendations have been made to close any fascial defect greater than or equal to 10 mm. Reported here is a case of herniation through a 3-mm trocar site incision and the discovery of a pre-existing fascial defect. CASE REPORT: A 32-year-old female underwent an uncomplicated laparoscopic tubal ligation using a 3-mm umbilical port. Prior to umbilical trocar removal at the completion of the case, the carbon dioxide was evacuated from the abdomen and the sleeve was withdrawn under direct vision. Neither the fascial nor skin incisions were sutured. On postoperative day two, the patient returned with omentum herniating from the 3-mm site. At surgery, a 1.5-cm pre-existing fascial defect was discovered adjacent to the trocar site. The hernia sac tracked laterally to the base of the umbilicus, and the omentum had slid into the sac and out the skin opening. CONCLUSION: As this report illustrates, herniation associated with laparoscopic trocar sites can occur with incisions as small as 3 mm. The presence of pre-existing fascial defects can cause increased morbidity in any laparoscopic surgery, and as illustrated in this report, may predispose the patient to site herniation. The detection and management of these defects is crucial in preventing postlaparoscopic complications.

Matrix metalloproteinase-1 and skin ageing in smokers.

Smokers look older than non-smokers of the same age. We have compared the concentrations of mRNA for matrix metalloproteinase 1 (MMP-1) in the buttock skin of smokers and non-smokers with quantitative real-time polymerase chain reactions. MMP-1 degrades collagen, which accounts for at least 70% of the dry weight of dermis. We report significantly more MMP-1 mRNA in the skin of smokers than non-smokers whereas no difference was seen for the tissue inhibitor of metalloproteinases 1 (TIMP-1) or the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase). We suggest that smoking-induced MMP-1 might be important in the skin-ageing effects of tobacco smoking.

Comparative quantification of IL-1beta, IL-10, IL-10r, TNFalpha and IL-7 mRNA levels in UV-irradiated human skin in vivo.

OBJECTIVE AND DESIGN: Ultraviolet (UV) exposure induces local immunosuppression and inflammation in human skin. Cytokines are, in part, responsible for these responses. To investigate the effects of UV-induced gene expression at the molecular level we established a sensitive in vivo/ex vivo method for a comparative quantification of cytokines and receptors involved in the local skin immune reactions. MATERIAL AND METHODS: Specific mRNA levels of human UV-irradiated skin were determined by real time quantification (TaqMan RT-PCR). Highly efficient PCR-reaction conditions were obtained by designing very short PCR-templates (72-87 bp). The most sensitive PCR-conditions were obtained by optimisation of primer and Mn(OAc)2-concentrations, which led to significant PCR signals (C(T)-value) of less than 36 cycles. A strong correlation between PCR efficiency of the internal control (GAPDH) compared to targets (IL-1beta, IL-10, IL-10r, TNFalpha, IL-7) allowed the use of deltadelta C(T)-method to quantify comparable mRNA levels. RESULTS: Interleukin-1beta (IL-1beta), Interleukin-10 (IL-10), and tumour necrosis factor alpha (TNFalpha) mRNA levels were increased in a time- and dose-dependent manner. Interleukin-1beta induction reached a maximum (approx. 44-fold) 6 h after a UV-dose equivalent to 3 times the minimal erythemal doses just perceptible (MEDjp). Maximal TNFalpha mRNA expression (approx. 14-fold) was also detected 6 h after UV exposure. Interleukin-10 mRNA induction reached a maximum of approximately 14-fold 24 h after UV-irradiation of 3 MEDjp. Time- and dose-dependent changes in Interleukin-7 and Interleukin-10 receptor mRNA levels did not occur after UV-irradiation. CONCLUSIONS: Time-distinct gene induction of IL-1beta, TNFalpha and IL-1beta is involved in UV-induced immune reactions, but no considerable changes were found for IL-10r or IL-7.

Excision of specific DNA-sequences from integrated retroviral vectors via site-specific recombination.

Vectors for gene transfer and gene therapy were developed which combine the advantages of the integrase and recombinase systems. This was achieved by inserting two loxP sites for specific DNA excision into an MESV based retroviral vector. We show that this 'retroviral lox system' allows the infection of cells and the expression of transferred genes. In addition, we constructed an efficient retrovirus-based expression system for a modified Cre recombinase. Functional tests for DNA excision from integrated retroviral lox vectors were performed by the use of a negative selectable marker gene (thymidine kinase). Cre expression in cells infected with retroviral lox vectors and subsequent BrdU selection for cells in which site-specific recombination has occurred results in large numbers of independent cell clones. These results were confirmed by detailed molecular analysis. In addition we developed retroviral suicide vectors in which the enhancer/promoter elements of both LTRs were replaced by lox sequences. We show that lox-sequences located in the LTRs of retroviral vectors are stable during retroviral replication. Potential applications of this system would be the establishment of revertants of retrovirus-infected cells by controlled excision of nearly the complete proviral DNA.

A two-component regulatory system for self/non-self recognition in Ustilago maydis.

In U. maydis the multiallelic b locus controls sexual and pathogenic development. In the b locus a gene coding for a regulatory protein had been identified, and it was suggested that the interaction of two b polypeptides specified by different alleles programs sexual development in this fungus. We now demonstrate the existence of a second regulatory gene in the b locus. We term this gene bW and refer to the former as the bE gene. Both genes exist in many alleles. Although unrelated in primary sequence, both genes are similar in their overall organization. The gene products display allele-specific variability in their N-terminal domains, show a high degree of sequence conservation in the C-terminal domains, and contain a homeodomain-related motif. Genetic evidence is provided to show that the pair of bE and bW polypeptides encoded by different b alleles is the key regulatory species.