[Analysis on treatment of eight extremely severe burn patients in August 2nd Kunshan factory aluminum dust explosion accident].

Objective: To summarize the measures and experience of treatment in mass extremely severe burn patients. Methods: The clinical data and treatment of 8 extremely severe burn patients in August 2 Kunshan factory aluminum dust explosion accident who were admitted in the 100th Hospital of PLA on August 2nd, 2014, were retrospectively analyzed. There were 4 males and 4 females, aging 22-45 (34±7) years, with total burn area of 55%-98% [(89±15)%] total body surface area (TBSA) and full-thickness burn area of 45%-97% [(80±21)%] TBSA. All the 8 patients were accompanied with severe shock, inhalation injury, and blast injury. According to the requirements of former PLA General Logistics Department and Nanjing Military Command, a treatment team was set up including a special medical unit and a special care unit, with Chai Jiake from the First Affiliated Hospital of PLA General Hospital as the team leader, Zheng Qingyi from the 175th Hospital of PLA (the Affiliated Dongnan Hospital of Xiamen University) as the deputy leader, the 100th Hospital of PLA as the treatment base, and burn care, respiratory, nephrology, nursing specialists from the First Affiliated Hospital of PLA General Hospital, and the burn care experts and nursing staff from the 180th Hospital of PLA, 118th Hospital of PLA, 98th Hospital of PLA, and 175th Hospital of PLA, and nurses from the 85th Hospital of PLA, 455th Hospital of PLA, 101th Hospital of PLA, 113th Hospital of PLA as team members. Treatment strategies were adopted as unified coordination by the superior, unified responsibility of team leader, division of labor and cooperation between team members, and multidisciplinary cooperation led by department of burns. With exception of one patient who received deep vein catheterization before admission, the other 7 patients were treated with deep vein catheterization 0.5 to 3.0 hours after admission to correct hypovolemic shock as soon as possible. Eight patients received tracheotomy, and 7 patients were treated with mechanical ventilation by ventilator in protective ventilation strategy with low tide volume and low volume pressure to assist breathing. Fiberoptic bronchoscopy was done one to three times for all the 8 patients to confirm airway injuries and healing status. Escharectomy and Meek dermatoplasty in the extremities of all the 8 patients were performed 3 to 6 days after injury for the first time. Escharectomy, microskin grafting, and covering of large pieces of allogeneic skin on the trunks of 4 patients were performed 11 to 16 days after injury for the second time. The broad-spectrum antibiotics were uniformly used at first time of anti-infective therapy, and then the antibiotics species were adjusted in time. The balance of internal environment was maintained and the visceral functions were protected. One special care unit was on responsibility of only one patient. Psychological intervention was performed on admission. The rehabilitative treatment was started at early stage and in company with the whole treatment. Results: Acute renal injury occurred in 5 patients within 36 hours after injury and their renal function was restored to normal 4 days after injury due to active adjustment of fluid resuscitation program. No pulmonary complications, such as severe pulmonary infection and ventilator-associated pneumonia, occurred in the survived patients. One of the 8 patients died, and the other 7 patients were cured successfully. The wounds were basically healed in 2 patients in 26 or 27 days by 2 or 3 times of operation, and in 5 patients by 4 or 5 times of operation. The basic wound healing time was 26-64 (48±15) days for all the 7 patients. Conclusions: Treatment strategies of unified coordination by the superior, unified responsibility of team leader, division of labor and cooperation between team members, and multidisciplinary cooperation led by department of burns are the bases to successful treatment. Correcting shock as soon as possible is the prerequisite and closing wound as soon as possible is the key to successful treatment. Comprehensive treatment measures, such as maintaining and regulating the function of viscera, improving the body immunity, and preventing and treating the complications, are the important components to successful treatment. It is emphasized that in the treatment of mass extremely severe burn patients, specialist burn treatment should always be in the dominant position, and other related disciplines may play a part in auxiliary function.

[Establishment and evaluation of a new method for determining hemodynamics of pulmonary hypertension rats].

Objective: By evaluating the hemodynamic parameters such as cardiac output (CO), right ventricular pressure (RVP), pulmonary artery pressure (PAP) and total pulmonary resistance index (TPRI) in pulmonary hypertension rat model, we established a more comprehensive hemodynamic evaluation system, which objectively evaluated the severity of disease and exercise tolerance in rats with pulmonary hypertension. Methods: SD rats were randomly divided into a control group and a model group with 5 rats in each group. The model group was intraperitoneally injected with SU5416 (20 mg/kg) and placed in an oxygen chamber at a 10% oxygen concentration for 21 days and then placed in a normoxic environment for 14 days. After modeling, rats were anesthetized and mechanically ventilated. The operator cut the skin along the right paraxial line, detached and ligated the intercostal artery, and then cut off the 3 and 4 ribs, exposing the heart and freeing aortic root about 0.2 cm. The flowmeter probe was set in the dissected aortic segment, and real-time recording time, blood flow waveforms, cardiac output were calculated accordingly. Then the needle attached to the baroreceptor was inserted into the right ventricle and the system acquired the right ventricular time-pressure waveform. After the waveform stabilized for about 30 seconds, the end of the cannula was sent to the pulmonary artery trunk through the entrance of the pulmonary artery to record the time-pressure curve of the pulmonary artery. Results: RVSP, PASP, PADP and mPAP in the model group were significantly higher than those of the control group [ RVSP(23.4±5.4) mmHg, 1 mmHg=0.133 kPa vs (56.4±13.0) mmHg, PASP (22.8±4.4) mmHg vs (58.5±14.9) mmHg, PADP (9.7±1.9) mmHg vs (30.3±7.0) mmHg, mPAP (14.1±2.7) mmHg vs (41.9±8.0) mmHg, all P<0.05 ]. Compared with the control group, the cardiac index in the model group was significantly lower [ CI (0.54±0.08) ml·min(-1)·g(-1) vs (0.40±0.09) ml·min(-1)·g(-1,) P=0.02 ]. Furthermore, compared with the control group, pulmonary vascular resistance index was significantly increased in the model group[PVRI (0.27±0.03) mmHg·ml(-1)·min(-1)·kg(-1) vs (0.06±0.01) mmHg·ml(-1)·min(-1)·kg(-1,) P<0.05]. The pathological results also showed that the middle part of pulmonary arterioles in the model group had muscular hypertrophy and muscular pulmonary arterioles, and even plexiform lesions. Conclusion: In this study, we established a new method that simultaneously determined several hemodynamic parameters such as RVSP, PASP, PADP, CO, CI and PVRI, which provided a more comprehensive assessment of hemodynamic changes in pulmonary hypertension rat models.

Tauroursodeoxycholic acid prevents hearing loss and hair cell death in Cdh23(erl/erl) mice.

Sensorineural hearing loss has long been the subject of experimental and clinical research for many years. The recently identified novel mutation of the Cadherin23 (Cdh23) gene, Cdh23(erl/erl), was proven to be a mouse model of human autosomal recessive nonsyndromic deafness (DFNB12). Tauroursodeoxycholic acid (TUDCA), a taurine-conjugated bile acid, has been used in experimental research and clinical applications related to liver disease, diabetes, neurodegenerative diseases, and other diseases associated with apoptosis. Because hair cell apoptosis was implied to be the cellular mechanism leading to hearing loss in Cdh23(erl/erl) mice (erl mice), this study investigated TUDCA's otoprotective effects in erl mice: preventing hearing impairment and protecting against hair cell death. Our results showed that systemic treatment with TUDCA significantly alleviated hearing loss and suppressed hair cell death in erl mice. Additionally, TUDCA inhibited apoptotic genes and caspase-3 activation in erl mouse cochleae. The data suggest that TUDCA could be a potential therapeutic agent for human DFNB12.

Otoprotective effects of erythropoietin on Cdh23erl/erl mice.

The Cdh23(erl/erl) mice are a novel mouse model for DFNB12 and are characterized by progressive hearing loss. In this study, erythropoietin (EPO) was given to the Cdh23(erl/erl) mice by intraperitoneal injection every other day from P7 for 7 weeks. Phosphate-buffered saline-treated or untreated Cdh23(erl/erl) mice were used as controls. Auditory-evoked brainstem response (ABR) thresholds and distortion product oto-acoustic emission (DPOAE) were measured in the mouse groups at the age of 4, 6 and 8 weeks. The results show that EPO can significantly decrease the ABR thresholds in the Cdh23(erl/erl) mice as compared with those of the untreated mice at stimulus frequencies of click, 8-, 16- and 32-kHz at three time points. Meanwhile, DPOAE amplitudes in the EPO-treated Cdh23(erl/erl) mouse group were significantly higher than those of the untreated groups at f2 frequency of 15383 Hz at the three time points. Furthermore, the mean percentage of outer hair cell loss at middle through basal turns of cochleae was significantly lower in EPO-treated Cdh23(erl/erl) mice than in the untreated mice (P<0.05). This is the first report that EPO acts as an otoprotectant in a DFNB12 mouse model with progressive hearing loss.

A new mouse mutant of the Cdh23 gene with early-onset hearing loss facilitates evaluation of otoprotection drugs.

We report a novel mutation (erlong, erl) of the cadherin 23 (Cdh23) gene in a mouse model for DFNB12 characterized by progressive hearing loss beginning from postnatal day 27 (P27). Genetic and sequencing analysis revealed a 208 T >C transition causing an amino-acid substitution (70S-P). Caspase expression was upregulated in mutant inner ears. Hearing was preserved (up to 35-dB improvement) in pan-caspase inhibitor Z-VAD-FMK-treated mutants compared with untreated mutants (P<0.05). Outer hair cell (OHC) loss in the cochleae of Z-VAD-FMK-treated mutants was significantly reduced compared with those of untreated mice. Thus, the erl mutation can lead to hearing loss through apoptosis. This is the first genetic mouse model of hearing loss shown to respond to otoprotective drug therapy. The short interval from initial hearing loss to deafness (P27-P90) makes this model ideal for screening and validating otoprotective drugs.

Hyperactivity, startle reactivity and cell-proliferation deficits are resistant to chronic lithium treatment in adult Nr2e1(frc/frc) mice.

The NR2E1 region on Chromosome 6q21-22 has been repeatedly linked to bipolar disorder (BP) and NR2E1 has been associated with BP, and more specifically bipolar I disorder (BPI). In addition, patient sequencing has shown an enrichment of rare candidate-regulatory variants. Interestingly, mice carrying either spontaneous (Nr2e1(frc) ) or targeted (Tlx(-) ) deletions of Nr2e1 (here collectively known as Nr2e1-null) show similar neurological and behavioral anomalies, including hypoplasia of the cerebrum, reduced neural stem cell proliferation, extreme aggression and deficits in fear conditioning; these are the traits that have been observed in some patients with BP. Thus, NR2E1 is a positional and functional candidate for a role in BP. However, no Nr2e1-null mice have been fully evaluated for behaviors used to model BP in rodents or pharmacological responses to drugs effective in treating BP symptoms. In this study we examine Nr2e1(frc/frc) mice, homozygous for the spontaneous deletion, for abnormalities in activity, learning and information processing, and cell proliferation; these are the phenotypes that are either affected in patients with BP or commonly assessed in rodent models of BP. The effect of lithium, a drug used to treat BP, was also evaluated for its ability to attenuate Nr2e1(frc/frc) behavioral and neural stem cell-proliferation phenotypes. We show for the first time that Nr2e1-null mice exhibit extreme hyperactivity in the open field as early as postnatal day 18 and in the home cage, deficits in open-field habituation and passive avoidance, and surprisingly, an absence of acoustic startle. We observed a reduction in neural stem/progenitor cell proliferation in Nr2e1(frc/frc) mice, similar to that seen in other Nr2e1-null strains. These behavioral and cell-proliferation phenotypes were resistant to chronic-adult-lithium treatment. Thus, Nr2e1(frc/frc) mice exhibit behavioral traits used to model BP in rodents, but our results do not support Nr2e1(frc/frc) mice as pharmacological models for BP.

A new spontaneous mutation in the mouse protocadherin 15 gene.

We have characterized a new allele of the protocadherin 15 gene (designatedPcdh15(av-6J)) that arose as a spontaneous, recessive mutation in the C57BL/6J inbred strain at Jackson Laboratory. Analysis revealed an inframe deletion in Pcdh15, which is predicted to result in partial deletion of cadherin domain (domain 9) in Pcdh15. Morphologic study revealed normal to moderately defective cochlear hair cell stereocilia in Pcdh15(av-6J) mutants at postnatal day 2 (P2). Stereocilia abnormalities were consistently present at P5 and P10. Degenerative changes including loss of inner and outer hair cells were seen at P20, with severe sensory cell loss in all cochlear turns occurring by P40. The hair cell phenotype observed in the 6J allele between P0 and P20 is the least severe phenotype yet observed in Pcdh15 alleles. However, young Pcdh15(av-6J) mice are unresponsive to auditory stimulation and show circling behavior indicative of vestibular dysfunction. Since these animals show severe functional deficits but have relatively mild stereocilia defects at a young age they may provide an appropriate model to test for a direct role of Pcdh15 in mechanotransduction.

Alms1-disrupted mice recapitulate human Alström syndrome.

Mutations in the human ALMS1 gene cause Alström syndrome (AS), a progressive disease characterized by neurosensory deficits and by metabolic defects including childhood obesity, hyperinsulinemia and Type 2 diabetes. Other features that are more variable in expressivity include dilated cardiomyopathy, hypertriglyceridemia, hypercholesterolemia, scoliosis, developmental delay and pulmonary and urological dysfunctions. ALMS1 encodes a ubiquitously expressed protein of unknown function. To obtain an animal model in which the etiology of the observed pathologies could be further studied, we generated a mouse model using an Alms1 gene-trapped ES cell line. Alms1-/- mice develop features similar to patients with AS, including obesity, hypogonadism, hyperinsulinemia, retinal dysfunction and late-onset hearing loss. Insulin resistance and increased body weight are apparent between 8 and 12 weeks of age, with hyperglycemia manifesting at approximately 16 weeks of age. In addition, Alms1-/- mice have normal hearing until 8 months of age, after which they display abnormal auditory brainstem responses. Diminished cone ERG b-wave response is observed early, followed by the degeneration of photoreceptor cells. Electron microscopy revealed accumulation of intracellular vesicles in the inner segments of photoreceptors, whereas immunohistochemical analysis showed mislocalization of rhodopsin to the outer nuclear layer. These findings suggest that ALMS1 has a role in intracellular trafficking.

The Mass1frings mutation underlies early onset hearing impairment in BUB/BnJ mice, a model for the auditory pathology of Usher syndrome IIC.

The human ortholog of the gene responsible for audiogenic seizure susceptibility in Frings and BUB/BnJ mice (mouse gene symbol Mass1) recently was shown to underlie Usher syndrome type IIC (USH2C). Here we report that the Mass1frings mutation is responsible for the early onset hearing impairment of BUB/BnJ mice. We found highly significant linkage of Mass1 with ABR threshold variation among mice from two backcrosses involving BUB/BnJ mice with mice of strains CAST/EiJ and MOLD/RkJ. We also show an additive effect of the Cdh23 locus in modulating the progression of hearing loss in backcross mice. Together, these two loci account for more than 70% of the total ABR threshold variation among the backcross mice at all ages. The modifying effect of the strain-specific Cdh23ahl variant may account for the hearing and audiogenic seizure differences observed between Frings and BUB/BnJ mice, which share the Mass1frings mutation. During postnatal cochlear development in BUB/BnJ mice, stereocilia bundles develop abnormally and remain immature and splayed into adulthood, corresponding with the early onset hearing impairment associated with Mass1frings. Progressive base-apex hair cell degeneration occurs at older ages, corresponding with the age-related hearing loss associated with Cdh23ahl. The molecular basis and pathophysiology of hearing loss suggest BUB/BnJ and Frings mice as models to study cellular and molecular mechanisms underlying USH2C auditory pathology.

Characterization of the triterpene saponins of the roots and rhizomes of blue cohosh (Caulophyllum thalictroides).

Seven triterpene saponins were isolated from n-butanol fractions of blue cohosh (Caulophyllum thalictroides) roots and rhizomes. Their structures were established by spectral ((1)H NMR, (13)C NMR, 2D-NMR, and APCI-MS) techniques and chemical reactions as hederagenin 3-O-alpha-L-arabinopyranoside (1); caulophyllogenin 3-O-alpha-L-arabinopyranoside (2); hederagenin 3-O-beta-D-glucopyranosyl-(1-->2)-alpha-L-arabinopyranoside (3); 3-O-alpha-L-arabinopyranosyl-hederagenin 28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranoside (4); 3-O-alpha-L-arabinopyranosyl- caulophyllogenin 28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranoside (5); 3-O-beta-D-glucopyranosyl-(1-->2)-alpha-L-arabinopyranosyl- echinocystic acid 28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranoside (6); 3-O-beta-D-glucopyranosyl-(1-->2)-alpha-L-arabinopyranosyl-hederagenin 28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranoside (7). All seven compounds were identified in this species for the first time.

Oxidative stress in aging in the C57B16/J mouse cochlea.

Presbycusis is a complex of high frequency hearing loss and disproportionate loss of speech discrimination that is seen concomitantly with physical signs of aging. Among the most extensively characterized strains of mice that show an early hearing loss is the C57B16/J strain, a strain that shows early onset of high frequency hearing loss at age 6 months and complete hearing loss by 1 year of age. The histopathology of this strain consists of loss of hair cells and spiral ganglion neurons in the basal turn, with a progression of loss of hair cells and ganglion neurons towards the apical portion of the cochlea as the animal ages. The process of aging has been extensively studied and although details differ in various organisms the consensus today is that oxidative stress, i.e. free radical-mediated tissue damage, is one of the core mechanisms of aging. Aerobic metabolism results in the creation of hydrogen peroxide and reactive oxygen species. These are normally detoxified by a variety of enzymes and free radical scavengers, including superoxide dismutase (SOD), catalase and glutathione. To determine whether oxidative stress plays a role in the pathophysiology of hearing loss in this mouse model of presbycusis we determined the relative change in mRNA production for selected free radical detoxifying enzymes in the C57B16/J mouse cochlea. Using semi-quantitative RT-PCR with tubulin mRNA as a control, relative levels of antioxidant enzyme mRNAs were determined. There was an overall increase in SOD1 mRNA levels when comparing 1 and 9 month time points, and a transient increase in the expression level of catalase mRNA. B6.CAST+ Ahl mice, which carry the C57B16/J genome but receive their Ahl gene from CAST mice, do not show these alteractions in antioxidant enzyme production. Our results suggest that at an age of 9 months, at which point significant hearing loss has developed, the C57B16/J mouse cochlea is exposed to increased levels of free radicals and that the Ahl gene of the C57B16/J mouse mediates this decrease in protective enzymes and therefore increase in levels of oxidative stress.

Hearing loss associated with the modifier of deaf waddler (mdfw) locus corresponds with age-related hearing loss in 12 inbred strains of mice.

The modifier of deaf waddler (mdfw) and age-related hearing loss (Ahl) loci were both discovered as inbred strain polymorphisms that affect hearing loss in mice. Both loci map to the same position on chromosome (Chr) 10. The mdfw locus interacts epistatically with the deaf waddler (dfw) mutation on Chr 6, and the Ahl locus is a major contributor to AHL in several inbred strains. To investigate the possibility of allelism, we examined the correspondence of mdfw and Ahl phenotypes among 12 inbred mouse strains. The effects of strain-specific mdfw alleles on hearing loss were assessed in dfw2J/+ F1 hybrids produced from mating BALB-dfw2J/+ mice with mice from each of 12 inbred strains. F1 hybrids were then assessed for hearing by auditory-evoked brainstem response threshold analysis and classified as dfw2J/+ or +/+ by polymerase chain reaction typing. Heterozygosity for dfw2J accelerated hearing loss in F1 hybrids derived from all strains tested, except those produced with the B6.CAST + Ahl congenic strain. dfw2J/+ F1 hybrids derived from parental strains 129P1/ReJ, A/J, BUB/BnJ, C57BR/cdJ, DBA/2J, NOD/LtJ and SKH2/J exhibited a severe hearing loss by 12 weeks of age. Those derived from strains 129T2/SvEmsJ, C3H/HeJ, CBA/CaJ and NON/LtJ exhibited only a slight to intermediate hearing loss at that age. The hearing loss associated with these strain-specific mdfw alleles corresponds with previously determined Ahl allele effects, providing additional evidence that mdfw and Ahl are manifestations of the same gene. A functional relationship therefore may exist between the Ca2+ transporting activity of the dfw gene (Atp2b2) and AHL.

A nuclear-mitochondrial DNA interaction affecting hearing impairment in mice.

The pathophysiologic pathways and clinical expression of mitochondrial DNA (mtDNA) mutations are not well understood. This is mainly the result of the heteroplasmic nature of most pathogenic mtDNA mutations and of the absence of clinically relevant animal models with mtDNA mutations. mtDNA mutations predisposing to hearing impairment in humans are generally homoplasmic, yet some individuals with these mutations have severe hearing loss, whereas their maternal relatives with the identical mtDNA mutation have normal hearing. Epidemiologic, biochemical and genetic data indicate that nuclear genes are often the main determinants of these differences in phenotype. To identify a mouse model for maternally inherited hearing loss, we screened reciprocal backcrosses of three inbred mouse strains, A/J, NOD/LtJ and SKH2/J, with age-related hearing loss (AHL). In the (A/J x CAST/Ei) x A/J backcross, mtDNA derived from the A/J strain exerted a significant detrimental effect on hearing when compared with mtDNA from the CAST/Ei strain. This effect was not seen in the (NOD/LtJ x CAST/Ei) x NOD/LtJ and (SKH2/J x CAST/Ei) x SKH2/J backcrosses. Genotyping revealed that this effect was seen only in mice homozygous for the A/J allele at the Ahl locus on mouse chromosome 10. Sequencing of the mitochondrial genome in the three inbred strains revealed a single nucleotide insertion in the tRNA-Arg gene (mt-Tr) as the probable mediator of the mitochondrial effect. This is the first mouse model with a naturally occurring mtDNA mutation affecting a clinical phenotype, and it provides an experimental model to dissect the pathophysiologic processes connecting mtDNA mutations to hearing loss.

A major gene affecting age-related hearing loss is common to at least ten inbred strains of mice.

Inbred strains of mice offer promising models for understanding the genetic basis of human presbycusis or age-related hearing loss (AHL). We previously mapped a major gene affecting AHL in C57BL/6J mice. Here, we show that the same Chromosome 10 gene (Ahl) is a major contributor to AHL in nine other inbred mouse strains-129P1/ReJ, A/J, BALB/cByJ, BUB/BnJ, C57BR/cdJ, DBA/2J, NOD/LtJ, SKH2/J, and STOCK760. F1 hybrids between each of these inbred strains and the normal-hearing inbred strain CAST/Ei retain good hearing, indicating that inheritance of AHL is recessive. To follow segregation of hearing loss, F1 hybrids were backcrossed to the parental strains with AHL. Auditory-evoked brain-stem response thresholds were used to assess hearing in more than 1500 N2 mice and analyzed as quantitative traits for linkage associations with Chromosome 10 markers. Highly significant linkage was found in all nine strain backcrosses, with the highest probability (LOD > 70) near the marker D10Mit112. This map position for Ahl is near the waltzer mutation (v) and the modifier of deaf waddler locus (mdfw), suggesting the possibility of allelism. Results from an intercross of C57BL/6J and NOD/LtJ mice indicate that the 6- to 10-month difference in AHL onset between these two strains is not due to allelic heterogeneity of the Ahl gene.

A new spontaneous mouse mutation in the Kcne1 gene.

A new mouse mutant, punk rocker (allele symbol Kcne1(pkr)), arose spontaneously on a C57BL/10J inbred strain background and is characterized by a distinctive head-tossing, circling, and ataxic phenotype. It is also profoundly and bilaterally deaf. The mutation resides in the Kcne1 gene on Chromosome (Chr) 16 and has been identified as a single base change within the coding region of the third exon. The C to T nucleotide substitution causes an arginine to be altered to a termination codon at amino acid position 67, and predictably this will result in a significantly truncated protein product. The Kcne1(pkr) mutant represents the first spontaneous mouse model for the human disorder, Jervell and Lange-Nielsen syndrome, associated with mutations in the homologous KCNE1 gene on human Chr 21.

Mouse mutants from chemically mutagenized embryonic stem cells.

The drive to characterize functions of human genes on a global scale has stimulated interest in large-scale generation of mouse mutants. Conventional germ-cell mutagenesis with N-ethyl-N-nitrosourea (ENU) is compromised by an inability to monitor mutation efficiency, strain and interlocus variation in mutation induction, and extensive husbandry requirements. To overcome these obstacles and develop new methods for generating mouse mutants, we devised protocols to generate germline chimaeric mice from embryonic stem (ES) cells heavily mutagenized with ethylmethanesulphonate (EMS). Germline chimaeras were derived from cultures that underwent a mutation rate of up to 1 in 1,200 at the Hprt locus (encoding hypoxanthine guanine phosphoribosyl transferase). The spectrum of mutations induced by EMS and the frameshift mutagen ICR191 was consistent with that observed in other mammalian cells. Chimaeras derived from ES cells treated with EMS transmitted mutations affecting several processes, including limb development, hair growth, hearing and gametogenesis. This technology affords several advantages over traditional mutagenesis, including the ability to conduct shortened breeding schemes and to screen for mutant phenotypes directly in ES cells or their differentiated derivatives.

Effect of a lipidic extract from lepidium meyenii on sexual behavior in mice and rats.

OBJECTIVES: To determine the effect of oral administration of a purified lipidic extract from Lepidium meyenii (MacaPure M-01 and M-02) on the number of complete intromissions and mating in normal mice, and on the latent period of erection (LPE) in rats with erectile dysfunction. METHODS: Mice and rats were randomly divided into several experimental and control groups. A 10% ethanol suspension of M-01 and M-02 was orally administered for 22 days to the experimental groups according to the dosage specified by the experimental design. On day 22, 30 minutes after the dose was administered to the male mice, 2 virgin female mice were placed with 1 male mouse. The number of complete intromissions of each male mouse in 3 hours was recorded. In an assessment of 1 day of mating, each male mouse was cohabited with 5 estrous female mice overnight. The number of sperm-positive females was recorded. The LPE was measured to assess the sexual function in rats with erectile dysfunction. By using a YSD-4G multifunction instrument, an electric pulse at 20 V was applied to stimulate the rat's penis, and the duration from the start of the stimulus to full erection was measured in seconds as the LPE. RESULTS: In the normal male mice, the number of complete intromissions during the 3-hour period was 16.33 +/- 1.78, 46.67 +/- 2.39, and 67.01 +/- 2.55 for the control group, M-01 group, and M-02 group, respectively. In the assessment of mating, the number of sperm-positive females increased from 0.6 +/- 0.7 in the control group to 1.5 +/- 0.5 in the M-01 experimental group. The LPE of male rats with erectile dysfunction was 112 +/- 13 seconds with a regular diet (control group). The oral administration of M-01 at a dose of 180 or 1800 mg/kg body weight and M-02 at a dose of 45, 180, or 1800 mg/kg body weight reduced the LPE to 54 +/- 12 seconds, 54 +/- 13 seconds, 71 +/- 12 seconds, 73 +/- 12 seconds, and 41 +/- 13 seconds, respectively. The LPE of the surgical rats treated with M-01 at the lowest dose (45 mg/kg) was 121 +/- 12 seconds; thus, the change was not significant. CONCLUSIONS: Oral administration of M-01 and M-02 enhanced the sexual function of the mice and rats, as evidenced by an increase in the number of complete intromissions and the number of sperm-positive females in normal mice, and a decrease in the LPE in male rats with erectile dysfunction. The present study reveals for the first time an aphrodisiac activity of L. meyenii, an Andean Mountain herb.