A missense mutation in the BRCA2 gene in three siblings with ovarian cancer.

Inherited susceptibility to ovarian cancer has been associated with germline defects at several loci. The major known ovarian cancer susceptibility gene is BRCA1 on chromosome 17q, which confers a risk of approximately 60% by the age of 70 years. Truncating mutations in BRCA2 on chromosome 13q also predispose to ovarian cancer, although they confer a lower risk than mutations in BRCA1. We have studied the molecular basis of ovarian cancer predisposition in a Finnish family with three affected sisters. Analysis of polymorphic markers provided evidence against linkage to BRCA1, but the sibship was consistent with linkage to BRCA2. Conformation-sensitive gel electrophoresis was used to screen the entire coding sequence of BRCA2. A G to A transition at nucleotide 8702 was observed, which is predicted to convert glycine 2901 to aspartate in the encoded protein. This sequence variant was not detected in 220 cancer-free Finnish control individuals, or in several hundred cancer families of many nationalities previously screened for BRCA2 mutations. Taken together with the fact that this amino acid residue and the surrounding region of BRCA2 is identical in mouse and chicken, the data suggest that this alteration is a disease-causing BRCA2 missense mutation. Previously published data indicate that the risks of breast and ovarian cancer conferred by BRCA2-truncating mutations varies with the position of the mutation in the gene. The missense mutation reported here suggests that the BRCA2 domain including and surrounding glycine 2901 may be more important in preventing neoplastic transformation in ovarian epithelium than in breast epithelium.     

Semen quality and associated reproductive indicators in middle-aged males: the role of non-malignant prostate conditions and genital tract inflammation

Purpose

To compare the associations between non-malignant prostate conditions, genital tract inflammation, and reproductive function in middle-aged men.

Methods

Three-hundred and eighty-two voluntary male subjects who underwent the screening for prostate health were recruited for the study. Semen quality and associated reproductive indicators, seminal inflammation, and prostate-related pathologies were evaluated.

Results

Sperm motility and prostate-related parameters were significantly impaired in patients with chronic prostatitis syndromes and lower urinary tract symptoms in comparison with controls. Elevated seminal markers of inflammation were in positive association with body mass index, prostate-specific antigen, and estradiol level in serum while in negative association with semen volume, total sperm count, and sperm motility. According to WHO reference limits, speculative cutoff values for WBC and IL-6 in semen to detect reduced sperm parameters were 0.342 M/mL and 56.8 ng/L, respectively.

Conclusions

According to our data, one of the possible pathways for impaired reproductive quality in male subjects >45 years could be related to infection and inflammation in the genital tract with subsequent (partial) obstruction and damage of prostate and other male accessory glands.     

Titanium wound chambers for wound healing research

Standardized and reproducible animal models are crucial in medical research. Rodents are commonly used in wound healing studies since, they are easily available, affordable and simple to handle and house. However, the most significant limitation of rodent models is that the wounds heal by contraction while in humans the primary mechanisms of healing are reepithelialization and granulation tissue formation. The robust contraction results in faster wound closure that complicates the reproducibility of rodent studies in clinical trials. We have developed a titanium wound chamber for rodent wound healing research. The chamber is engineered from two pieces of titanium and is placed transcutaneously on the dorsum of a rodent. The chamber inhibits wound contraction and provides a means for controlled monitoring and sampling of the wound environment in vivo with minimal foreign body reaction. This technical report introduces two modalities utilizing the titanium chambers in rats: (1) Wound in a skin island model and, (2) Wound without skin model. Here, we demonstrate in rats how the “wound in a skin island model” slows down wound contraction and how the “wound without skin” model completely prevents the closure. The titanium wound chamber provides a reproducible standardized models for wound healing research in rodents.     

The effects of intentional cryoablation and radio frequency ablation of renal tissue involving the collecting system in a porcine model

PURPOSE: Ablative techniques for the treatment of urological malignancy are gaining acceptance and they are likely to become more widely used in clinical practice. Indications and limitations of the technologies are still evolving. In a porcine model we evaluated the safety and efficacy of cryotherapy and radio frequency ablation (RFA) of cortical and deep renal tissue. MATERIALS AND METHODS: In 11 swine argon gas based cryoablation or RFA of renal tissue adjacent to the collecting system was performed using a laparoscopic or percutaneous approach. Lesions created in renal units 30 days or 2 hours prior to harvest were termed chronic or acute. Using single or multiple 17 gauge cryoneedles or 3.0 mm cryoprobes and 2 freeze-thaw cycles (10-minute freeze and 5-minute thaw) 13 acute and 10 chronic cryolesions were made. Using a single 16 gauge umbrella-shaped RFA probe and 2 heating cycles to maximum impedance 13 acute and 4 chronic RFA lesions were made. Gross and microscopic tissue analysis was performed to assess lesion size and renal parenchymal, collecting system and arterial effects. Acute cryolesion size estimation by laparoscopic or transcutaneous ultrasound (US) was compared with pathological lesion size. RESULTS: Acute cryolesions on hematoxylin and eosin staining demonstrated uniform coagulative necrosis of renal parenchyma and chronic cryolesions demonstrated uniform necrosis with fibrous scar formation. Interlobar artery (adjacent to renal pyramid) preservation occurred in 7 of 13 acute and 5 of 9 chronic cryolesions. Urothelial architecture was preserved in 8 of 13 acute and 7 of 9 chronic cryolesions. Acute and chronic RFA lesions demonstrated indeterminate necrosis on hematoxylin and eosin staining, although triphenyl tetrazolium chloride staining of gross specimens confirmed necrosis most definitively in renal cortex. Interlobar artery preservation occurred in 6 of 13 acute and 3 of 4 chronic RFA lesions. Urothelial architecture was preserved in 1 of 13 acute and 2 of 4 chronic RFA lesions. Acute cryolesion dimensions measured by laparoscopic US equaled or underestimated lesion size measured grossly in all 6 cases. Lesion dimensions measured by transcutaneous US equaled or underestimated true lesion size in 3 of 6 cases. In 3 of 6 lesions transcutaneous US overestimated true lesion size by 20%, 76% and 260%, respectively. CONCLUSIONS: Renal cortical tissue can be effectively destroyed by cryoablation or RFA. However, treatment of deep parenchymal lesions with either modality may result in incomplete ablation. Cryosurgery but not RFA spares the collecting system in an acute setting. However, healing or regrowth of the urothelium may occur with time after RFA. Laparoscopic US is more accurate for cryolesion monitoring than transcutaneous US.     

Functional role of P-glycoprotein in the human blood-placental barrier

OBJECTIVE: In vitro and animal experiments suggest that P-glycoprotein forms a functional barrier between maternal and fetal blood circulation in the placenta, thus protecting the fetus from exposure to xenobiotics during pregnancy. In this study we aimed to characterize the role of P-glycoprotein in the blood-placental barrier by use of dually perfused human placenta. METHODS: Twenty-eight human placentas were obtained after delivery, and both the maternal side and the fetal side were perfused for 2 hours. Saquinavir was used as a probe drug for P-glycoprotein-dependent active transfer, and PSC833 (valspodar) or GG918 was used as an inhibitor of P-glycoprotein function in a maternal-to-fetal and fetal-to-maternal perfusion setting. Genotyping for ABCB1 (C3435T and G2677A/T) polymorphism and quantification of P-glycoprotein expression were done for each placenta. RESULTS: The fetal-to-maternal transfer of saquinavir was 108-fold higher (P = .003) compared with transfer from the maternal to the fetal direction. Preperfusion with PSC833 increased the placental transfer of saquinavir by 7.9-fold (P < .001), and preperfusion with GG918 increased it by 6.2-fold (P < .001). The end-perfusion transfer (percentage) of saquinavir at 120 minutes was 11-fold (P < .001) and 6-fold (P < .001) higher in placentas preperfused with PSC833 and GG918, respectively, compared with control. However, PSC833 had no effect on the transfer of saquinavir from the fetal to the maternal direction (P = .79). P-glycoprotein expression was correlated with the PSC833-induced change in the saquinavir transfer (r = 0.75, P = .086). ABCB1 polymorphism did not affect the PSC833- or GG918-induced change in the saquinavir transfer. CONCLUSIONS: P-glycoprotein has a major functional role in the human blood-placental barrier but a negligible role in the removal of substances from the fetal circulation to maternal blood. Pharmacologic blockade of P-glycoprotein function can lead to disruption of the blood-placental barrier and increase the transfer of P-glycoprotein substrates to the fetal side by several-fold, which may be a noteworthy mechanism for teratogenicity.     

Reactions of N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil) with 2'-deoxyadenosine

N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil, 1; 0.6 mM) was allowed to react with 2'-deoxyadenosine (16.1 mM) at physiological pH (cacodylic acid, 50% base), and the reactions were followed by HPLC-MS and HPLC-MS/MS techniques. Although the predominant reaction observed was chlorambucil hydrolysis, ca. 7% of 1 reacted with various heteroatoms of the nucleoside. The principal site of alkylation was N1. Several other adducts were also detected. The N1, N6, N3, and N7 derivatives were characterized by means of MS/MS, UV, and (1)H NMR. The N6 adduct is derived directly from alkylation of N6 of 2'-dAdo. Dimroth rearrangement of the N1 adduct to the N6 adduct was very slow under the reaction conditions employed. Minor adducts such as a carbohydrate derivative were tentatively characterized by MS/MS. No cross-links were detected. The role of chlorambucil-2'-deoxyadenosine adducts in the cytotoxicity and mutagenicity of 1 is also discussed.