p62 improves AD-like pathology by increasing autophagy.

The multifunctional protein p62 is associated with neuropathological inclusions in several neurodegenerative disorders, including frontotemporal lobar degeneration, amyotrophic lateral sclerosis and Alzheimer's disease (AD). Strong evidence shows that in AD, p62 immunoreactivity is associated with neurofibrillary tangles and is involved in tau degradation. However, it remains to be determined whether p62 also plays a role in regulating amyloid-ß (Aß) aggregation and degradation. Using a gene therapy approach, here we show that increasing brain p62 expression rescues cognitive deficits in APP/PS1 mice, a widely used animal model of AD. The cognitive improvement was associated with a decrease in Aß levels and plaque load. Using complementary genetic and pharmacologic approaches, we found that the p62-mediated changes in Aß were due to an increase in autophagy. To this end, we showed that removing the LC3-interacting region of p62, which facilitates p62-mediated selective autophagy, or blocking autophagy with a pharmacological inhibitor, was sufficient to prevent the decrease in Aß. Overall, we believe these data provide the first direct in vivo evidence showing that p62 regulates Aß turnover.

Clusterin overexpression in both malignant and nonmalignant prostate epithelial cells induces cell cycle arrest and apoptosis.

Expression of the castration-induced clusterin protein is incompatible with the survival of human prostate cancer cells in tissues and in cell culture. To investigate the fate of human prostate epithelial cells, when engineered to maintain expression of clusterin protein, we have used an IRES-hyg vector and hygromycin selection. PC-3 prostate tumour cells were substantially more sensitive to clusterin expression than nonmalignant PNT1a cells, showing multiple phenotypic changes including cell cycle arrest and increased apoptosis. The results strengthen the hypothesis that clusterin expression is proapoptotic. Expression of exogenous clusterin in both cell types resulted in its relocation from the cytoplasm and a nuclear accumulation of the protein, as was also seen in the same cells when apoptosis was induced by etoposide treatment. To survive clusterin expression, the PC-3 tumour cells developed apoptosis-inhibitory properties. This could have significance for the resistance of prostate cancers to chemo/radiotherapy, where clusterin overexpression is observed.

Nuclear translocation of a clusterin isoform is associated with induction of anoikis in SV40-immortalized human prostate epithelial cells.

Clusterin gene expression is potently induced in experimental models in which apoptosis is activated, such as rat prostate involution following castration. Nevertheless, its precise physiological role has not yet been established, and both anti-apoptotic and pro-apoptotic functions have been suggested for this gene. Clusterin expression level depends on cell proliferation state, and we recently showed that its over-expression inhibited cell cycle progression of SV40-immortalized human prostate epithelial cells PNT2 and PNT1a. Here we studied clusterin expression in PNT1a cells subjected to serum-starvation with the aim of defining clusterin early molecular changes following apoptosis induction. Under serum-starvation conditions, decreased growth rate, slow rounding-up of cells, cell detachment, and formation of apoptotic bodies indicative of anoikis (detachment-induced apoptosis) were preceded by significant downregulation of 70 kDa clusterin precursor and upregulation of 45-40 kDa isoforms. On the 8th day of serum-free culturing, only the higher molecular weight protein-band of about 45 kDa was clearly induced and accumulated in detached cells and apoptotic bodies in which PARP was activated. Anoikis was preceded by induction and transloction of a 45-kDa clusterin isoform to the nucleus. Thus, nuclear targeting of a specific 45-kDa isoform of clusterin appeared to be an early and specific molecular signal triggering anoikis-death. Considering also that clusterin is downregulated during prostate cancer onset and progression, and that its upregulation has inhibited DNA synthesis and cell cycle progression of immortalized human prostate epithelial cells, we suggest that clusterin might be a new anti-oncogene in the prostate.

The effect of heat shock on amino acid transport and cell volume in 3T3 cells.

In 3T3 cells temperatures higher than physiological stimulated amino acid transport activity in a dose-dependent manner up to 44 degrees C. However, the temperature increase did not induce widespread transport increase of all other nutrients tested. The activities of both amino acid transport systems A and ASC were enhanced within a few minutes following cell exposure to increased temperature. The maintenance of this effect required continuous exposure of the cells to hyperthermia. Kinetic analysis indicated that the stimulation of the activity of transport System A occurred through a mechanism affecting Vmax rather than Km. The continuous presence of cycloheximide did not prevent the transport changes induced by hyperthermia. These results suggest that the increased amino acid uptake reflects an activation or relocation of existing amino acid transport proteins. During the hyperthermic treatment, the content of ninhydrin-positive substances (NPS), mostly amino acids, increased within the cells and the accumulation of these compatible osmolytes was parallelled by an increase in cell volume. The withdrawal of amino acids from the culture medium immediately before and during the shock phase counteracted the increase and reduced the NPS content but did not prevent the increase in amino acid transport, the cell swelling and the induction of the heat shock response.

Osmotic regulation of ATA2 mRNA expression and amino acid transport System A activity.

When porcine endothelial cells were exposed to hypertonicity, both the level of ATA2 (amino acid transporter 2) mRNA and activity of amino acid transport System A increased transiently, peaking after about 6 and 9 h, respectively. Cycloheximide, like actinomycin D, prevented both responses, showing that an earlier step also involves protein synthesis. Withdrawal of hypertonicity after 6 h increased the rate of down regulation. These findings confirm that ATA2 is a major isoform of System A and show that changes in the expression of ATA2 mRNA precede both the induction and subsequent down regulation of transport activity.

Induction of BGT-1 and amino acid system A transport activities in endothelial cells exposed to hyperosmolarity.

We studied the responses to hypertonicity of cultured endothelial cells from swine pulmonary arteries. In 0.5 osmol/kgH(2)O medium, initial cell shrinkage was followed by a regulatory volume increase (RVI), complete after 1 h, concomitant with an increase in cellular K(+) content. Then the activity of amino acid transport System A increased, accompanied by an accumulation of ninhydrin-positive solutes (NPS), reaching a peak at approximately 6 h. The subsequent decline in System A activity was paralleled by an induction of the betaine-GABA transporter (BGT-1), detected as increases of BGT-1 mRNA and of transport activity, which peaked at approximately 24 h. Inhibitors of transcription or translation prevented induction of both transport activities. The increased expression of BGT-1, which involved activation of "tonicity-responsive enhancer," was inhibited by 5 mM extracellular betaine. Cellular K(+) concentration gradually declined after the accumulation of NPS and during the induction of BGT-1. This very effective adaptation to hypertonicity suggests it has a physiological role.

Enhanced quality of capacitated spermatozoa from oligoasthenozoospermic men after incubation in test yolk medium.

To improve artificially the quality of poor sperm samples to be used in assisted procreation procedures, Test Yolk Buffer (TYB) incubation effect was investigated. After adding the TYB procedure to the standard swim-up technique, a significant (P < 0.001) increase in spermatozoa velocity was observed, while the other seminal parameters were not affected. Three of 11 couples who had not had success after three intraperitoneal inseminations (IPI) with sperm prepared by the usual swim-up technique obtained pregnancies after IPI with TYB-incubated spermatozoa. These preliminary results suggest that TYB incubation for capacitated spermatozoa be employed in the assisted procreation strategies applied for male infertility.

Negative effect of stress of in vitro fertilization program on quality of semen.

There are controversial data on the effects of stress on semen quality during treatment for infertility. We have compared the parameters of semen obtained from 84 patients on the day of the ovocyte pick-up during an in vitro fertilization and embryo transfer program (IVF) with those of semen collected from the same patients on the day of admission to the program (up to 3 months earlier). Abnormal semen has been found in 2 of the 84 patients on the day of admission and in 11 on the day of IVF. There were no improvements in semen quality of any patient at the second test. All except two parameters evaluated objectively by the Hamilton-Thorn Motility analyzer were significantly worse (p < 0.001) on the day of IVF, the exceptions were the percentage with moderate or slow motility and the Linear Index. These data indicate that the emotional stress to a subject enrolled in an IVF program negatively affects the quality of semen. Some suggestions are made for improved management of these patients.

Computerized slow-staged freezing of semen from men with testicular tumors or Hodgkin's disease preserves sperm better than standard vapor freezing.

Two methods of freezing semen taken from patients with testicular tumors or Hodgkin's disease before treatment were compared. Many patients already have semen abnormalities, so an optimal method is extremely important. Ejaculates from 8 patients with testicular tumors and 20 with Hodgkin's disease were frozen by fast-freezing or by slow-staged freezing. Effects of motility, viability, and swelling after thawing were significantly impaired with both methods. However, cryosurvival was better after slow- than fast-freezing: motility 24% +/- 12.4% versus 15% +/- 11.2%; viability 24.1% +/- 11.4% versus 17.3% +/- 10.4%, swelling 33.3% +/- 11% versus 27.6% +/- 12.8%. The effects were equal for normal and abnormal sperm. Sperm from tumor patients should be frozen by slow-staged freezing method in spite of the higher cost and longer time.

IVF: induction of ovulation in poor responders.

All IVF programs have a consistent rate of failure in inducing ovulations. Pharmacological induction of ovulation is otherwise crucial for an IVF program because of the need for more than one ovum. Since it is well known that the best candidates for HMG treatment are hypogonadotropic women a short reversible hypogonadotropic state was induced in IVF patients by LH-RH agonist (Buserelin). Superovulation was then achieved with very high initial doses of FSH (Metrodin) in order to maximize the ovarian response. This technique used in 116 IVF women induced a satisfactory follicle growth even in 70% of the patients already poorly responsive to HMG stimulation.

Ovarian stimulation of IVF patients: effects of the reversible hypogonadotrophic state induced by GnRH agonist.

Multiple ovulation was induced in 122 hypogonadotrophic IVF patients with large doses of HMG. The hypogonadotrophic state, short and reversible, was obtained by nasal administration of a GnRH agonist (200 micrograms, five times per day). In the 97 induced cycles, a mean of 9.1 follicles was recorded. A comparison of the results obtained for 36 patients who had already been treated with clomiphene and HMG showed both significantly more follicles per cycle (8.5 versus 3.0) and an increase in oocytes retrieved (6.7 versus 1.3) when treated with the agonist and HMG. In addition 11 of 18 already poorly responsive patients had normal responses. The luteal phase was supported by either HCG or progesterone injection. Plasma progesterone profiles were satisfactory and, as expected, the highest progesterone concentrations were associated with HCG treatment.

Long term effects of cryoapheresis and cytostatic treatment in essential mixed cryoglobulinemia.

We studied the effects of cryoapheresis combined with different immunosuppressive treatments on the course of the glomerulonephritis of essential mixed cryoglobulinemia. The study was carried out on 11 patients. The effects of immunosuppressive treatments on cryoglobulin rebound after cryoapheresis varied widely. In those responding with sustained reduction in serum cryoglobulin levels, creatinine clearance increased, an effect that lasted several years in 4 patients. In one patient cryoglobulin disappeared, with almost fully recovery of renal function and normalization of blood pressure. One patient died of acute liver failure shortly after the first observation and another entered regular dialysis treatment. All the other patients are still alive after follow-up of 2-9 years. These results compare favourably with those reported by other investigators and suggest that cryoapheresis and cytostatic drugs are beneficial for glomerulonephritis associated with essential mixed cryoglobulinemia.

Suppression of post-apheresis autoantibody rebound in cryoglobulinemia and cold agglutinin hemolytic anemia.

We studied the effect of immunosuppressive treatment on the autoantibody rebound that follows autoantibody withdrawal accomplished by extracorporeal plasma treatment. The study was carried out on 9 patients, 8 having essential mixed cryoglobulinemia and 1 cold agglutinin hemolytic anemia. As immunosuppressive drugs we used either cyclophosphamide and prednisolone or arabinoside C and cyclophosphamide. The effect of immunosuppressive treatment on autoantibody rebound (Igm anti-IgG; IgM cold agglutinins) varied widely, a suppressive effect of varying degree having been observed in about half the cases treated with either drug regimen.

Glomerular deposits of rheumatoid factor in glomerulonephritis.

One hundred and forty-four kidney biopsy specimens with various forms of glomerulonephritis were studied to assess the presence of Rheumatoid Factor (RF) deposits. RF deposits were found in 21 specimens: six with acute post-streptococcal glomerulonephritis, two with crescentic glomerulonephritis, four with lupus nephritis, eight with essential mixed cryoglobulinaemia glomerulonephritis, and one with end-stage kidney disease. Blocking and elution studies carried out on specimens with essential mixed cryoglobulinaemia provided evidence that the RF deposits derive from circulating monoclonal RF. This data suggests that RF participates in the formation of glomerular immune deposits in several forms of immune complex mediated glomerulonephritis.

Glomerular localization of circulating antiglobulin activity in essential mixed cryoglobulinemia with glomerulonephritis.

Kidney biopsy samples from 27 patients with essential mixed cryoglobulinemia of the IgG-IgM(k) type and glomerulonephritis were studied to assess whether glomerular immunodeposits display antiglobulin (AG) activity similar to that of serum cryo-IgM. A preparation of heat-aggregated human IgG (FAIgG) was used to search for tissue AG activity, and blocking tests and reactivity tests were carried out to define the nature of this activity. Glomerular localization of FAIgG was observed in 17 out of 27 kidney specimens, the positive findings being always associated with IgM deposits. Prior exposure of tissue sections to anti-IgM serum blocked the FAIgG reaction, but no such effect was produced by the pretreatment with other antisera. The positive FAIgG tissue specimens yielded a similar fluorescence pattern with aggregated alkylated-reduced IgG, but did not react at all with the aggregated F(ab')2 or aggregated albumin. The IgM recovered in the eluate of a kidney biopsy specimen displayed AG activity. Patients with AG deposits showed more severe histologic changes and a greater renal functional impairment than did those without. The data support the notion that circulating cryo-IgM anti-IgG participates in the formation of glomerular immunodeposits and in the genesis of renal damage.

Preparation of a partially desialylated human chorionic gonadotrophin (hCG) and its use for induction of ovulation after ovarian stimulation with human menopausal gonadotrophin.

A method for the large scale preparation of partially desialylated human chorionic gonadotrophin suitable for human use is reported. To obtain the desired grade of desialylation and to avoid the presence of the enzyme in the modified hormone, neuraminidase coupled to Sepharose 4B was used. The preparation showed to be active in vitro (OAAD and SVW tests) and its half-life was found to be 13 min in the rat and 75 min in human beings. This desialo hCG proved to be effective in inducing ovulation in amenorrhoeic women. Among 39 induced cycles 31 ovulations and 5 pregnancies occurred.

Effect of three serotonin antagonists on plasma prolactin response to suckling in puerperal women.

The effect of the acute administration of three serotonin antagonists on plasma PRL levels and on the PRL response to suckling was investigated in a group of puerperal women. A single oral dose of metergoline or methysergide induced a significant decrease of plasma PRL levels and abolished the PRL response to suckling. Cyproheptadine administration did not modify either the plasma PRL levels or the PRL response to suckling. These results are discussed in light of the known pharmacological properties of the three antiserotonergic drugs.