Non-viral transfection of goat germline stem cells by nucleofection results in production of transgenic sperm after germ cell transplantation.

Germline stem cells (GSCs) can be used for large animal transgenesis, in which GSCs that are genetically manipulated in vitro are transplanted into a recipient testis to generate donor-derived transgenic sperm. The objectives of this study were to explore a non-viral approach for transgene delivery into goat GSCs and to investigate the efficiency of nucleofection in producing transgenic sperm. Four recipient goats received fractionated irradiation at 8 weeks of age to deplete endogenous GSCs. Germ cell transplantations were performed 8-9 weeks post-irradiation. Donor cells were collected from testes of 9-week-old goats, enriched for GSCs by Staput velocity sedimentation, and transfected by nucleofection with a transgene construct harboring the human growth hormone gene under the control of the goat beta-casein promoter (GBC) and a chicken beta-globin insulator (CBGI) sequence upstream of the promoter. For each recipient, transfected cells from 10 nucleofection reactions were pooled, mixed with non-transfected cells to a total of 1.5 × 10(8) cells in 3 ml, and transplanted into one testis (n = 4 recipients) by ultrasound-guided cannulation of the rete testis. The second testis of each recipient was removed. Semen was collected, starting at 9 months after transplantation, for a period of over a year (a total of 62 ejaculates from four recipients). Nested genomic PCR for hGH and CBGI sequences demonstrated that 31.3% ± 12.6% of ejaculates were positive for both hGH and CBGI. This study provides proof-of-concept that non-viral transfection (nucleofection) of primary goat germ cells followed by germ cell transplantation results in transgene transmission to sperm in recipient goats.

Improved survival from diagnosis of AIDS in adult cases in the United Kingdom and bias due to reporting delays.

OBJECTIVE: To measure developments in survival patterns among United Kingdom adult AIDS cases. DESIGN: A follow-up survey of cases reported voluntarily to the national surveillance schemes was undertaken to obtain up-to-date information on vital status. METHODS: All reporting clinicians who had a current AIDS patient not known to have died whose AIDS-defining illness was diagnosed before the end of September 1990 were contacted. A total of 3984 cases were included in the analysis. RESULTS: An extra third of deaths other than those reported through routine channels were ascertained by follow-up. Median survival for patients diagnosed before and after the end of 1986 increased from 15 to 18 months for men who had sex with men presenting with Kaposi's sarcoma, from 10 to 19 months for other men who had sex with men and from 7 to 16 months for all others. Improvement in survival was greatest in the first 3 months. One-third of patients have been surviving 2 years or more. Factors observed with independent effects on improved survival are recent diagnosis, younger age and larger cumulative AIDS case load of reporting centre. HIV encephalopathy and other central nervous system symptoms may be associated with poorer survival. CONCLUSIONS: Survival patterns have been changing and generally improving. Average survival for very recent cohorts tends to be underestimated because longer survival has been observed in patients for whom there is a longer delay between AIDS diagnosis and report to the Communicable Disease Surveillance Centre. Information on mortality is improved by active follow-up.

United Kingdom AIDS survival in adults.

OBJECTIVE: To examine the impact of age, year and region of AIDS diagnosis on early (up to 12 months) and late survival of AIDS patients in different exposure categories; to describe the hazard pattern from 12 months after AIDS diagnosis. PATIENTS: A total of 4577 UK adult AIDS diagnoses to the end of 1991 in men who have sex with men, 273 AIDS cases in injecting drug users, 411 AIDS patients infected by blood products, and 535 other adult AIDS cases, mainly ascribed to heterosexual transmission. Deaths have been recorded for 4739 of these 5796 AIDS patients. RESULTS: The influence of calendar year and region of AIDS diagnosis on survival were short-term, for the most part operative only within the first year of follow-up. The monthly death-rate was roughly constant from 12 to 48 months post-AIDS [pooled estimate, 0.055 with 95% confidence interval (CI), 0.053-0.057] but was more than halved for 4-year survivors (pooled estimate, 0.022; 95% CI, 0.017-0.027). About 7% of AIDS cases diagnosed in 1990-1991 survive for at least 48 months. Survival after AIDS diagnosis shortens with advancing age at AIDS diagnosis: the relative hazard per decade of age (1.35; 95% CI, 1.29-1.41 in the first year after AIDS) is significantly greater (P < 0.001) in the first year after AIDS diagnosis than from 12 to 48 months (1.19; 95% CI, 1.13-1.25 in the second epoch). CONCLUSIONS: The influence of covariates, including age, is strongest in the first year of follow-up after AIDS diagnosis. Monthly death-rate is roughly constant at 0.055 from 12 to 48 months post-AIDS and at 0.022 thereafter.

The cloning of the human follicle stimulating hormone receptor and its expression in COS-7, CHO, and Y-1 cells.

Follicle stimulating hormone (FSH) receptor clones were isolated from a human testis cDNA library. Characterization of the cDNA clones showed that the DNA and predicted amino acid sequences of the long open reading frame differed from a previously published human ovarian FSH receptor sequence (Minegish et al. (1991) Biochem. Biophys. Res. Commun. 175, 1125-1130) by seven nucleotides and five amino acids. A human FSH receptor splice variant was also identified and characterized. A full-length human FSH receptor cDNA was engineered for expression in COS-7, CHO, and Y-1 cells. In transient transfections of COS-7 cells and stable transfections of Y-1 cells, efficient FSH receptor mRNA accumulation and isolation of FSH-responsive cell lines occurred only when an intron was included in the 5' untranslated region of the FSH receptor transcription unit. Y-1 cells stably transfected with the FSH receptor responded to FSH treatment by rounding up and by synthesizing increased amounts of progesterone. Stably transfected CHO cell lines, which responded to FSH by synthesizing increased amounts of cAMP, were isolated irrespective of the presence of the heterologous intron. The FSH-responsive CHO and Y-1 cell lines may be suitable for the development of better in vitro FSH bioassays. These cells also constitute a convenient source of human FSH receptor protein for use in radioreceptor assays and in studies of receptor-ligand interactions.

Type IV collagen in Hodgkin's disease. An immunohistochemical study.

A series of 50 lymph nodes affected by Hodgkin's disease (HD) and 10 further nodes exhibiting the features of reactive follicular hyperplasia (RFH) have been studied. A peroxidase-antiperoxidase (PAP) sequence for the demonstration of type IV collagen has been applied to routinely processed paraffin-embedded sections of these specimens. Blood vascular structures of all calibers were clearly demonstrated, as were lymphatic sinuses. Structures recognizable as the latter were generally lacking in HD, but the sinus structures in the cases of RFH were well defined. Blood vessels were highly active on type IV collagen staining in the interfollicular areas of reactive nodes but were relatively scanty in lymphocyte-predominant, mixed-cellularity, and lymphocyte-depleted HD. However, in the nodular sclerosing Rye subtype, blood vessels containing type IV collagen were as numerous as in RFH and could be seen within and around the cellular nodules. A similar number of type IV collagen-containing blood vessels were also apparent in the cellular variant of nodular sclerosing HD. The results are compared with those obtained on immunostaining for Factor VIII-related antigen and on binding of Ulex europaeus lectin I, and type IV collagen is considered to be the most sensitive vascular marker compared with these.

Repair of upper ureteric obstruction with Boari flap and psoas hitch.

A case of iatrogenic traumatic obstruction to both upper and lower ureters resulting in severe hydronephrosis is reported. Continuity and good functional result was achieved with a Boari flap and psoas hitch to the renal pelvis.

Cognitive exposure versus avoidance in patients with chronic pain: adherence matters.

BACKGROUND: Behavioural exposure methods can reduce pain-avoidance behaviours, but outcomes vary. One possible explanation is that patients employ cognitive (experiential) avoidance during behavioural exposure. If so, reducing cognitive avoidance during behavioural exposure should help. One option is interoceptive exposure (IE), which involves sustained exposure (via attention) to pain sensations. In order to test if IE could improve outcomes from behavioural exposure, this study with mixed chronic pain patients compared outcomes from a cognitive behavioural therapy (CBT) pain management programme incorporating either IE or distraction from pain. METHODS: One hundred forty chronic pain patients were randomly assigned to CBT + IE or CBT + distraction. Outcome measures included pain, disability, depression and medication. Measures reflecting degree of threat of pain were also employed (catastrophizing, fear-avoidance, pain self-efficacy and pain acceptance). An intention-to-treat approach, using mixed-effects model repeated measures, as well as conventional inferential statistical tests, effect sizes and reliable change indices were employed to evaluate the outcomes up to 1-year post-treatment. RESULTS: Significant improvements were achieved by both treatment conditions on all outcome measures and on measures reflecting the threatening nature of pain, with no differences between treatment conditions. CONCLUSIONS: The addition of IE to behavioural exposure did not improve outcomes. However, higher adherence to either attentional strategy was associated with larger effect sizes on all measures, suggesting factors shared by the two treatments could have contributed to the outcomes. Taken as a whole, the results suggest that increasing adherence to treatment strategies, possibly by motivational measures, would improve the overall outcomes of these interventions.

Is adherence to pain self-management strategies associated with improved pain, depression and disability in those with disabling chronic pain?

There is generally good evidence that pain management interventions that include self-management strategies can substantially reduce disability and improve psychological well-being in patients with chronic pain. Reductions in unhelpful responses, especially catastrophising and fear-avoidance beliefs, have been established as key contributors to these gains. In contrast, there is surprisingly little evidence that adherence to self-management strategies contributes to achieving these outcomes. Difficulties in defining and measuring the use of pain self-management strategies have been obstacles for this research. Using a pragmatic way of assessing the practice of specific strategies this study investigated their ability to account for changes in pain, disability and depressive symptoms after a 3-week cognitive-behavioural pain management program. The post-treatment outcomes on these dimensions were found to be statistically and, for many, clinically significant. Consistent with previous research, reductions in catastrophising and fear-avoidance beliefs, and increased pain self-efficacy beliefs, were also associated with these gains. But the key new finding was that there was a clear gradient between adherence to specific self-management strategies and reductions in pain, disability and depressive symptoms. Furthermore, adherence to the self-management strategies was predictive of better outcomes even after controlling for the moderating effects of initial catastrophising, fear-avoidance and pain self-efficacy beliefs.

Characterization of argininosuccinate lyase (EC 4.3.2.1) from Chlamydomonas reinhardtii.

We have isolated and characterized argininosuccinate lyase (ASL; EC 4.3.2.1) from the photosynthetic green alga, Chlamydomonas reinhardtii. The general properties of Chlamydomonas ASL are very similar to those described previously for ASLs from phylogenetically diverse organisms. The algal ASL has a native Mr, determined by gel-filtration chromatography, of 218,000 +/- 25,000, and a pI of 5.4-5.6. The Km for argininosuccinate at 37 degrees C and pH 7.5 is 0.26 mM. The subunit Mr of Chlamydomonas ASL is approx. 50,000, determined by SDS/polyacrylamide-gel electrophoresis, in contrast with a previously reported value of 39,000. Rabbit antisera prepared against the Mr-50,000 protein completely abolished ASL activity in vitro. In contrast, serum prepared against the Mr-39,000 protein was ineffective in inhibiting ASL activity. Despite the general similarity of the physical properties of Chlamydomonas ASL and those of other ASLs, antiserum raised against the algal ASL did not cross-react with ASL preparations from Escherichia coli, Saccharomyces cerevisiae or bovine liver.

Antarctic fish tubulins: heterogeneity, structure, amino acid compositions and charge.

1. Tubulins purified from the brain tissues of three Antarctic fishes (Notothenia gibberifrons, Notothenia coriiceps neglecta, and Chaenocephalus aceratus) contain equimolar quantities of the alpha and beta chains and are free of microtubule-associated proteins (MAPs) and other non-tubulin proteins. 2. When examined by isoelectric focusing and by two-dimensional electrophoresis, brain tubulins from the Antarctic fishes were found to be highly heterogeneous; each was resolved into 15-20 distinct variants. The range of isoelectric points displayed by the Antarctic fish tubulins (5.30-5.75) is slightly more basic than that of bovine brain tubulin (5.25-5.60). 3. Peptide mapping demonstrated that tubulins from the Antarctic fishes and the cow differ in structure. 4. The amino acid compositions of piscine and mammalian tubulins are similar, but the Antarctic fish tubulins apparently contain fewer glutamyl and/or glutaminyl residues than do tubulins from the temperate channel catfish (Ictalurus punctatus) and the cow. 5. Native tubulin from N. coriiceps neglecta possesses 1-2 fewer net negative charges per tubulin dimer than does bovine tubulin. 6. We suggest that the enhanced assembly of Antarctic fish tubulins at low temperatures (-2 to +2 degrees C) results from adaptive, perhaps subtle, changes in their tubulin subunits.

Heterogeneity and structure of brain tubulins from cold-adapted Antarctic fishes. Comparison to brain tubulins from a temperate fish and a mammal.

Tubulins purified from brain tissue of Antarctic fishes assemble in vitro to form microtubules at the low temperatures experienced by these extreme psychrophiles (Williams, R. C., Jr., Correia, J. J., and DeVries, A. L. (1985) Biochemistry 24, 2790-2798). We have initiated studies to determine the structural requirements for assembly of Antarctic fish tubulins at low temperatures. As a first step we have compared the heterogeneity, structures, amino acid compositions, and net charge of brain tubulins purified from three Antarctic fishes (Notothenia gibberifrons, Notothenia coriiceps neglecta, and Chaenocephalus aceratus), from the temperate channel catfish (Ictalurus punctatus), and from a mammal (the cow). Each preparation contained the alpha- and beta-tubulins and was free of microtubule-associated proteins. When examined by isoelectric focusing and by two-dimensional electrophoresis, brain tubulins from the Antarctic fishes were found to be highly heterogeneous; each was resolved into approximately 20 isoelectric variants. The distributions of the isotubulins from the cold-adapted fishes were similar but differed significantly from those of tubulins from catfish and cow. The average isoelectric points of the alpha- and beta-tubulins from the Antarctic fishes were more basic than the isoelectric points of the corresponding tubulins from bovine brain. Peptide mapping confirmed that tubulins from the Antarctic fishes and the mammal differed in structure. The amino acid compositions of fish and mammalian tubulins were similar, but Antarctic fish tubulins apparently contained fewer Glx residues than did catfish or bovine tubulins. Finally, native tubulins from an Antarctic fish and the cow differed slightly in net negative charge. Thus, brain tubulins from the cold-adapted fishes differ structurally from the tubulins of a temperate fish and of a mammal.

The impact of stimulus 'value' in infant novelty preference.

Various factors have been identified that influence the robust phenomenon of novelty preference in infants. The present study consisted of two experiments that investigated whether conditioned 'value' is another variable that influences infants' preferences. In Experiment 1, novelty preference was established to simple tones despite the tones differing only in frequency. In Experiment 2, novelty preference was manipulated by pairing a primary reinforcer with the familiarization tone such that the conditioned value overrode novelty preference. The findings raise questions about the universality of predicting infants' preferences solely on the foundation of amount of stimulus experience.

Routine surveillance data on AIDS and HIV infections in the UK: a description of the data available and their use for short-term planning.

In the UK surveillance of AIDS and HIV infection is based on routine reporting systems. Whilst attempts are made to ensure that AIDS data are as complete as possible, numbers of reports fluctuate from month to month for reasons which are described. In 1986 there was an increase in death certificates naming AIDS as a cause of death in patients who were not identifiable in the surveillance data. More active surveillance is now undertaken in order to minimize this and other possible discrepancies. It is probable that most cases of AIDS are reported and therefore these data can be used to describe trends in the epidemic by 'risk group'. Laboratory reports of HIV antibody-positive tests could give an earlier indication of trends because of the long incubation period of AIDS. But these laboratory data are difficult to interpret because they represent an incomplete and biased sample of all positive persons. AIDS cases are still being reported at a rate which is increasing approximately exponentially. Short-term predictions are presented showing a growth in the epidemic which is consistent with previously published predictions. Most cases are in the homosexual risk group. New asymptomatic homosexual patients with HIV antibody are still being identified. The epidemic of AIDS in haemophilia patients should be of finite size although new cases of AIDS are likely to continue to be diagnosed for several years. AIDS due to blood transfusion given in the UK before donor screening appears to be a much smaller epidemic. The epidemic in drug abusers is increasing. Heterosexually acquired AIDS and HIV infections are being reported in small but increasing numbers.

Effects of exercise on fetal-placental growth and uteroplacental blood flow in the rat.

The effects of daily maternal exercise on fetal-placental growth and development and the associated changes in uteroplacental blood flow were studied in pregnant rats. Pregnant females were exercised for 1 h (0% grade at 28 m/min) daily between either days 1 and 12, 1 and 18, 1 and 22, 12 and 18, or 12 and 22 of gestation and compared with nonexercised controls. Exercise between days 1 and 12 of pregnancy had no effect on fetal-placental parameters (i.e., fetal weight or gestation length) relative to controls. In contrast, exercise between days 1 and 22 and 12 and 22 resulted in a lower number of live pups born, an increase in gestational length, and an increased birth weight of the live pups relative to controls. Exercise between days 1 and 18 or 12 and 18 induced a significant (p less than or equal to 0.05) suppression of uteroplacental blood flow, the number of viable fetuses, and placental weights as compared with controls. Daily exercise had no effect on ovarian function as indicated by the comparable serum progesterone levels in all groups. These data indicate that an exercised-induced depression of uteroplacental blood flow is related to impaired fetal-placental growth and development during pregnancy in the rat.

Effects of alcohol on event-related brain potentials produced by viewing a simulated traffic signal.

The P-300 event-related brain potential was monitored during simulated traffic signal tasks for 10 subjects who were tested at three levels of mean blood alcohol content: 0.00%, 0.06%, and 0.13%. A significant increase in latency occurred for the 0.13% level indicating that an increased mental processing time was required to determine if a red or a green light had been presented.

Survival of adults with AIDS in the United Kingdom.

Accurate estimates of expected survival times and survival rates of AIDS patients are important both for estimating the prognosis of individuals and for monitoring the progress of the HIV/AIDS epidemic as new treatments are introduced. They are also needed for projecting future numbers of AIDS cases. Data on reported AIDS cases held at the PHLS AIDS Centre at the Communicable Disease Surveillance Centre and the Scottish Centre for Infection and Environmental Health confirmed the time, age, and reporting delay effects identified in earlier analyses of the United Kingdom AIDS database. The duration of survival after AIDS is diagnosed has improved since the epidemic began--median survival was 10.6 months in cases diagnosed before 1987 and has been at least 18.4 months in cases diagnosed each year since then. People who are diagnosed younger live longer--median survival fell from 21.6 months at age 15 to 29 to 12.6 months at age 45 or over. Delay in reporting AIDS cases adversely affects survival estimates for cases reported in recent years. Survival was longer in cases reported over a year after diagnosis of AIDS--23.7 months compared with 16.9 months in those reported less than a year after diagnosis. The experience of the hospital, measured by its cumulative AIDS caseload, was an important factor in the survival of men who have sex with men presenting with Kaposi's sarcoma alone or 'other' diagnoses--survival was shorter for cases reported from smaller centres. Men who have sex with men with Pneumocystis carinii pneumonia alone or other opportunistic infections alone who were known to be HIV positive before being diagnosed with AIDS had a shorter survival after being diagnosed than those who were unaware of their HIV infection. This supports the hypothesis that treatment for HIV infection and prophylaxis may extend the period before AIDS develops but reduce the period between developing AIDS and dying.