Mitochondrial DNA content and 4977 bp deletion in unfertilized oocytes

Previous studies analysing the incidences of mitochondrial DNA (mtDNA) deletions and mtDNA content in unfertilized oocytes in relation to donors' age have been controversial. The objective of the study was to compare these two parameters in unfertilized oocytes and relate them to the donors' age. Fifty-two women donated 155 unfertilized metaphase II (MII) oocytes. The incidence of 4977 bp deletion was 34.6%, and the mtDNA copy number was 598 350 +/- 265 862. Women >or=35 years of age had a significantly higher incidence of 4977 bp deletion, lower mtDNA copy number, higher FSH level and poorer ovarian response when compared with younger women. The mtDNA copy number was negatively correlated with the donor's age. The higher incidence of mtDNA deletion and lower mtDNA copy number in older women suggested that these two parameters may reflect ovarian ageing.     

Human papillomavirus typing with a polymerase chain reaction-based genotyping array compared with type-specific PCR

BACKGROUND: Type-specific persistence of human papillomavirus (HPV) infection can cause invasive cervical cancer. OBJECTIVES: To evaluate the efficacy of HPV detection and typing with a general polymerase chain reaction (PCR)-based genotyping array and to compare it with a type-specific PCR assay. STUDY DESIGN: Four hundred and thirty-three cervical samples were tested with a modified MY11/GP6+ PCR-based reverse-blot assay (EasyChip HPV Blot; King Car, Taiwan [hereafter HPV Blot]) and with 20 genotypes of L1-type-specific PCR (HPV-6, -11, -16, -18, -31, -33, -35, -39, -45, -51, -52, -53, -56, -58, -59, -62, -66, -68, -70, and -71 [CP8061]). RESULTS: The concordance of the two tests in determining HPV positivity was 96.8% (419/433), with a Cohen's kappa=0.93 (95% CI: 0.90-0.97) and McNemar's test of P=1.0, which indicates excellent agreement. The overall concordance of the two tests in the identification of type-specific HPV was 91.0% (394/433). Sensitivity (90-100%), specificity (99.2-100%), and accuracy (98.6-100%) rates of HPV Blot against the gold standard were satisfactory for HPV-16, -18, -58, -33, -52, -39, -45, -31, -51, -70 while HPV-71 (63.6%) had suboptimal sensitivity. Though the kappa values between the two tests for many individual genotypes could not be reliably calculated because of low positivity, the kappa values for HPV-16, -52, and -58 were excellent (0.93, 0.96, and 0.95, respectively). CONCLUSION: The modified MY11/GP6+ PCR-based HPV Blot assay is accurate and sensitive for detection and genotyping of HPV in cervical swab samples.     

Stability of simian virus 40 (SV40) mRNA species.

An anaerobic plating technique for bacteriophage is described. This technique results in larger plaque sizes for at least nine of the bacteriophages that grow on Salmonella and Escherichia coli. The advantage of this plating technique for the selection of nonsense mutants in certain phage strains is discussed.     

Severe classical congenital muscular dystrophy and merosin expression

Vajsar J, Chitayat D, Becker LE, Ho M, Ben-Zeev B, Jay V. Severe classical congenital muscular dystrophy and merosin expression. Clin Genet 1998: 54: 193–198. 0 Munksgaard, 1998
It has been suggested that patients with autosomal recessive merosin deficient congenital muscular dystrophy (CMD), as opposed to the merosin positive cases form a homogeneous subgroup of a clinically more severe form of CMD. We examined merosin expression in muscle biopsies from five children with the severe classical form of CMD. Merosin deficiency was found only in 1 patient, a 6–year-old female, with abnormal brain myelination. However, her initial biopsy did not reveal the classical picture of dystrophy. The four merosin positive cases exhibited severe muscle weakness but their brain imagings were normal. There were no familial cases, except for the mother of 1 patient who had a milder form of the disease, suggesting an autosomal dominant mode of inheritance.
In contrast to previous reports, the merosin deficient CMD cases were rare in our group. Furthermore, merosin positive cases were also associated with severe phenotype suggesting that a severe phenotype is not exclusive to merosin deficient cases. Finally, the absence of merosin in a neonate with hypotonia and weakness can be helpful in making a definitive diagnosis of CMD, even though the dystrophic process may not be evident yet and histology may be non-specific.     

Lymphomas of true histiocytic origin. Expression of different phenotypes in so-called true histiocytic lymphoma and malignant histiocytosis.

The authors determined the phenotypes of neoplastic cells in true histiocytic lymphoma and malignant histiocytosis by using a large panel of monoclonal antibodies and enzyme histochemistry procedures. Although the phenotypes overlapped slightly, the authors noted a distinct pattern in these tumors. The tumor cells of malignant histiocytosis generally expressed the monocyte markers CD11b, CD11c, CD14, and CD45, especially after induction with phorbol ester. In contrast, the tumor cells of true histiocytic lymphoma exhibited a marker expression very similar to that of Reed-Sternberg cells in Hodgkin's disease. These cells expressed markers CD30, 2H9, and 1A2, but rarely expressed CD11b, CD11c, CD14, or CD45. Regardless of their cytologic features, the tumor cells from both types of histiocytic lymphoma exhibited diffuse nonspecific esterase and acid phosphatase activities, and they expressed histiocyte markers CD15, CD68, LN5, 1E9, and M387 to varying degrees. The tumor cells from both lymphomas did not exhibit T- or B-cell markers, T-cell receptor or immunoglobulin gene rearrangements, or gene translation products, even when they were induced with phorbol ester. The phenotypic expression in these two histiocytic malignancies suggests that they are derived from different types of histiocytes, or from histiocytes in different stages of maturation or differentiation, or from histiocytes that have distinct mechanisms of tumorigenic transformation. The expression of circulating monocyte markers in malignant histiocytosis suggests that this tumor originates in monocytes or free histiocytes, whereas the phenotype of true histiocytic lymphoma is compatible with an origin in fixed histiocytes, which generally are devoid of the monocyte markers CD11b and CD14.     

Local properties of a functionally graded interphase between cementum and dentin

The study of natural interfaces may provide information necessary to engineer functionally graded biomaterials for bioengineering applications. In this study, the mechanical, structural, and chemical composition variations associated with a region between cementum and dentin were studied with the use of nanoindentation, microindentation, optical microscopy, and Raman microspectroscopy techniques. Three-millimeter-thick transverse sections (N = 5) were obtained from the apical one-third of the roots of sterilized human molars. The samples were ultrasectioned at room temperature with the use of a diamond knife and an ultramicrotome. Longitudinal ground sections of 100 microm thickness were prepared and stained with von Kossa stain to determine the mineralized regions within the molar roots. Raman microspectroscopy was used to determine the relative inorganic content, mainly apatite (PO4(3-)nu1 mode at 960 cm(-1)) and organic content, mainly collagen (C--H stretch at 2940 cm(-1)) between cementum and dentin bulk tissues. The microindentation and nanoindentation results indicated a gradual transition in hardness from cementum to dentin over a width ranging from 100 to 200 microm. However, the variation in hardness data for cementum and dentin by nanoindentation was larger (0.62 +/- 0.21, 0.77 +/- 0.14 GPa) than from microindentation (0.49 +/- 0.03, 0.69 +/- 0.07 GPa). Within the 100 to 200 microm region there was a 10 to 50 microm fibrillar hydrophilic cementum-dentin junction (CDJ) with mechanical properties significantly lower than either the cementum or the dentin side of CDJ. Light microscopy revealed a 100 to 200 microm translucent region between cementum and dentin. Raman microspectroscopy results showed a variation in organic and inorganic composition 80 to 140 microm wide. It was concluded that a morphologically and biomechanically different CDJ lies within a wider cementum-dentin interphase. Hence, cementum, dentin, and the interphase can be classified as a functionally graded dental tissue within the root of a tooth.     

Effects of the androgenic anabolic steroid, nandrolone decanoate, on adrenocorticotropin hormone, corticosterone and proopiomelanocortin, corticotropin releasing factor (CRF) and CRF receptor1 mRNA levels in the hypothalamus, pituitary and amygdala of the rat

There is increasing abuse of androgenic anabolic steroids (AAS) by non-athletes. AAS abuse has been associated with psychiatric symptoms such as mania, major depression and aggression and the development of dependence. Little is known about the effects of AAS on hypothalamic-pituitary-adrenal axis function or corticotropin releasing factor, which may be involved in mediating some of the psychiatric symptoms associated with AAS abuse. Male Sprague-Dawley rats received one daily intra-muscular injection of the AAS nandrolone decanoate (ND, 15 mg/kg) or vehicle for 3 days. Animals were sacrificed either 1 h or 24 h after the last injection, brain regions dissected and trunk blood collected. Corticotropin releasing factor (CRF), CRF receptor1 (CRF-R1) and proopiomelanocortin (POMC) mRNAs were measured with solution hybridization/RNase protection. Circulating levels of corticosterone and adrenocorticotropin hormone (ACTH) were determined using radioimmunoassays. One hour following the last injection, ND significantly increased circulating levels of both corticosterone and ACTH levels. In the amygdala, CRF mRNA levels were unchanged 1 h after the last injection of ND but were significantly reduced at 24 h. The same was found for hypothalamic POMC. No significant AAS effects were observed on: hypothalamic CRF mRNA; POMC mRNA in the amygdala or CRF R1 mRNA in the anterior pituitary.     

Immunosuppression in Kenyan visceral leishmaniasis

Cell-mediated immune responses were evaluated in 15 patients with active visceral leishmaniasis from Masinga location in eastern Kenya where the disease is endemic. Age and sex matched controls were selected from a village school in the same area. In vivo studies were carried out by skin testing with leishmanin, tuberculin, streptococcal and candida antigens. Lymphocyte blastogenic transformation to the mitogens phytohaemagglutinin (PHA) and concanavalin A (Con A) and the antigens purified protein derivative (PPD), streptokinase-streptodornase (SKSD) and leishmanial antigen (LA) was studied in vitro. The results showed that immunosuppression in visceral leishmaniasis in Kenya was both specific and non-specific. In the majority of patients there was complete anergy to all antigens in vivo and in vitro. The suppression of responses to mitogens was less marked. Recovery of non-specific responses preceded the development of specific immunity. In a small number of patients (23%) immune unresponsiveness to leishmanial antigens persisted 1 year after parasitological cure.     

Ultrastructure of preimplantation human embryos co-cultured with human ampullary cells

Ova with two pronuclei were co-cultured with established human ampullary cell lines and various stages of preimplantation embryonic development were monitored by Nomarski optics and then assessed by transmission electron microscopy (TEM). Fifteen embryos ranging from the 2-cell stage to blastocyst hatching were examined for normal and abnormal features. Their ultrastructure was similar to that of embryos cultured in Whittingham's T6 medium, reported previously. Seven embryos were evidently morphologically normal and showed good organization of fine structure. Most cellular organelles underwent progressive changes during early development. There was evidence of enhanced embryonic genome activation at the 8-cell stage. Invariably, all embryos had few too many fragments, some internalized, which were later segregated into the blastocoele or found outside the trophoblast of the late morula and blastocysts. Six grossly 'normal' embryos assessed by Nomarski had multiple nuclei of various dimensions, which highlights the subjectivity of embryo assessment in the IVF laboratory. Incomplete incorporation of chromatin into nuclei and formation of micronuclei were evident in some blastomeres. The results are discussed in relation to early embryonic loss, prevalent in IVF. Significant events reported include the detection of centrioles at the 8-cell stage, cavitation of the early blastocyst and the initiation of blastocyst hatching visualized by TEM.