Quantification of HIV DNA in the brain by PCR: differences between fresh frozen and formalin fixed tissue.

HIV-1 DNA extracted from frozen and formalin fixed brain tissue can be detected using PCR. This work has been extended by amplifying, using semiquantitative PCR, HIV DNA extracted from frontal lobe tissue of 16 patients with AIDS (eight positive and eight negative for p24 antigen). DNA was amplified using HIV-1 pol gene digoxigenin labelled primers and detected by chemiluminescence and densitometry. Cloned standards were amplified in parallel for quantification. HIV DNA levels detected in frozen tissue showed a correlation with p24 positivity and the severity of the histological diagnosis. This correlation was less clear in the formalin fixed material.     

Epidermal mosaicism and Blaschko''s lines.

To test the hypothesis that epidermal rather than dermal mosaicism determines Blaschko's lines in hypomelanosis of Ito (HI), we studied the distribution of chromosomal mosaicism in four patients. In two, mosaicism had not been detected in lymphocytes or dermal fibroblasts, but was clearly shown in epidermal keratinocytes; furthermore, the abnormal cell line was confirmed to the hypopigmented epidermis and the normal epidermis contained only normal cells. Negative findings in the other two patients might be because of mosaicism which was undetected either because it was submicroscopic or because it was present in melanocytes, which have not yet been studied. These preliminary results support the ideas that (1) Blaschko's lines represent single clones of epidermal cells; (2) in patients with HI and severe neurological involvement mosaicism, if detectable, is best shown in keratinocytes; and (3) the cytogenetic defect in epidermal cells may be directly responsible for the failure of pigmentation in HI.     

Y chromosome microdeletions, in azoospermic or near-azoospermic subjects, are located in the AZFc (DAZ) subregion

Submicroscopic deletions of the Y chromosome and polymorphisms of the androgen receptor (AR) gene in the X chromosome have been observed in men with defective spermatogenesis. To further define the subregions/genes in the Y chromosome causing male infertility and its relationship to polymorphisms of the AR polyglutamine tract, we screened the genomic DNA of 202 subfertile males and 101 healthy fertile controls of predominantly Chinese ethnic origin. Y microdeletions were examined with 16 sequence-tagged site (STS) probes, including the RBM and DAZ genes, spanning the AZFb and AZFc subregions of Yq11, and related to the size of trinucleotide repeat encoding the AR polyglutamine tract. Y microdeletions were detected and confirmed in three out of 44 (6.8%) of azoospermic and three out of 86 (3.5%) severely oligozoospermic patients. No deletions were detected in any of the patients with sperm counts of >0.5 x 10(6)/ml, nor in any of the 101 fertile controls. All six affected patients had almost contiguous Y microdeletions spanning the entire AZFc region including the DAZ gene. The AZFb region, containing the RBM1 gene, was intact in five of the six subjects. Y deletions were not found in those with long AR polyglutamine tracts. Our study, the first in a Chinese population, suggest a cause and effect relationship between Y microdeletions in the AZFc region (possibly DAZ), and azoospermia or near-azoospermia. Y microdeletions and long AR polyglutamine tracts appear to be independent contributors to male infertility.      

Dysferlin is a plasma membrane protein and is expressed early in human development

Recently, a single gene, DYSF, has been identified which is mutated in patients with limb-girdle muscular dystrophy type 2B (LGMD2B) and with Miyoshi myopathy (MM). This is of interest because these diseases have been considered as two distinct clinical conditions since different muscle groups are the initial targets. Dysferlin, the protein product of the gene, is a novel molecule without homology to any known mammalian protein. We have now raised a monoclonal antibody to dysferlin and report on the expression of this new protein: immunolabelling with the antibody (designated NCL-hamlet) demonstrated a polypeptide of approximately 230 kDa on western blots of skeletal muscle, with localization to the muscle fibre membrane by microscopy at both the light and electron microscopic level. A specific loss of dysferlin labelling was observed in patients with mutations in the LGMD2B/MM gene. Furthermore, patients with two different frameshifting mutations demonstrated very low levels of immunoreactive protein in a manner reminiscent of the dystrophin expressed in many Duchenne patients. Analysis of human fetal tissue showed that dysferlin was expressed at the earliest stages of development examined, at Carnegie stage 15 or 16 (embryonic age 5-6 weeks). Dysferlin is present, therefore, at a time when the limbs start to show regional differentiation. Lack of dysferlin at this critical time may contribute to the pattern of muscle involvement that develops later, with the onset of a muscular dystrophy primarily affecting proximal or distal muscles.     

The source and action of histamine in the isolated guinea-pig gallbladder

We have investigated the effects of histamine on motility of the gallbladder and characterized the receptor types involved. Histamine and the histamine H₁-receptor agonist, 2-thiazolylethylamine (2-TEA) contracted the isolated guinea-pig gallbladder strip in a dose dependent manner. The contractile response to histamine was shifted to the right by the H₁-receptor antagonist, mepyramine. In pre-contracted gallbladder strips, the H₂-receptor agonist dimaprit reduced the tension generated in a dose dependent fashion. The histamine H₂-receptor antagonist, ranitidine shifted the histamine concentration effect curve to the left and attenuated the dose dependent relaxations elicited at high concentrations. The histamine H₃-receptor agonist, (R)--methylhistamine (RMHA) elicited dose dependent contraction of the tissue which was significantly inhibited in the presence of mepyramine. The effects of electrical field stimulation (EFS) on the strips were not significantly altered by the presence of RMHA (10^–10–10^–7 M) indicating little pre-synaptic H₃ activity in this tissue. Histamine immunoreactivity (IR) was detected in gallbladder whole mount preparations of the mucosa and the muscularis/serosa. The histamine IR appeared cell bound in cells of varying morphological characteristics but no IR was detected in nerve fibres or cell bodies (ganglia). Alcian blue staining was consistent with the distribution of histamine IR cells as mast cells. The results indicate that histamine is distributed in the guinea-pig gallbladder and it can regulate contractile activity via activation of H₁ and H₂ but not H₃ receptors.     

Comparison of the in situ changes in lymphoid cells during infection with infectious bursal disease virus in chickens of different ages.

In situ immunocytochemical staining was used to characterise leukocyte changes and determine tropism of infectious bursal disease virus following infection of neonate and 3-week-old chickens. In the bursae of both groups, massive replication of the virus, a rapid depletion of B cells and an influx of CD4(+) TCR-alphabeta(1)(+) and CD8 (+) TCR-alphabeta(1)(+) cells was detected within 4 days post-inoculation. Leukocyte changes in the spleen, thymus and Harderian gland were similar in both groups. From 8 days post-inoculation onwards all the lymphoid organs became repopulated with leukocytes and tissue architecture began to be slowly restored. Virus neutralizing antibodies developed more slowly in neonate birds and at 21 days post-inoculation the titres were much lower compared to older birds. Lack of clinical signs in neonate chickens was neither due to a failure to respond to the virus, to recruit leukocytes to the infected tissues nor to a lack of viral replication.     

Persistence of free HBV DNA in body secretions and liver despite loss of serum HBV DNA after interferon-induced seroconversion

Following interferon therapy, a chronic hepatitis B (HBV) carrier lost all serum markers of active viral replication and became anti-HBe positive but remained positive for free and replicative HBV-DNA in semen, saliva, urine, and liver four months later. At 12 months, when he also developed anti-HBs, urine and saliva analysed for free HBV-DNA were positive. Despite histological remission and loss of HBV-DNA from serum, the potential for transmission of HBV and reactivation of disease remain.     

Developmentally programmed expression of AID in chicken B cells

In mice, activation induced deaminase, AID, is expressed only in germinal center B cells. It is required for the initiation of somatic hypermutation and class switch recombination. In chickens and most mammals immunoglobulin gene rearrangement generates limited diversity and the primary immunoglobulin repertoire depends on subsequent somatic hypermutation or gene conversion. Immunoglobulin gene conversion in chickens starts in the embryonic bursa, before antigen exposure. The demonstrated requirement for AID for gene conversion in the bursal lymphoma cell line, DT40, implies developmental regulation of AID expression. To test this prediction, we examined the timing and location of AID mRNA expression. An abrupt increase in AID mRNA coincided with the onset of extensive Ig gene conversion in the bursa. Expression was also detected at earlier stages, implying either that expression of AID is not the only controlling factor for gene conversion, or that gene conversion can precede the formation of bursal follicles.     

Hoechst staining and exposure to UV laser during flow cytometric sorting does not affect the frequency of detected endogenous DNA nicks in abnormal and normal human spermatozoa

Controlling the sex of offspring by the separation of X and Y chromosome-bearing spermatozoa using flow cytometry has been reported as a clinical technique aiding prevention of X-linked diseases. Although this technique has resulted in several hundred normal births in animals and at least one human birth, there is still concern over its genetic safety due to the involvement of two potentially mutagenic agents: UV light and the fluorochrome dye, Hoechst 33342 (H33342). Human spermatozoa, particularly those considered abnormal, may be more likely to suffer DNA damage following exposure to mutagenic agents, compared with other mammalian species. The stability of normal fresh and decondensed human spermatozoa were examined after exposure to a range of levels of UV and H33342 staining, using an assay that detects endogenous nicks in the DNA of spermatozoa. The stability of abnormal and normal, fresh and frozen-thawed human spermatozoa was examined following UV laser, H33342 staining and flow cytometry treatments utilizing the same assay. There was an increase in the presence of endogenous nicks when spermatozoa were decondensed compared with fresh spermatozoa. There was no increase in the incidence of nicks in any group of spermatozoa after UV and fluorochrome exposure compared with controls without exposure.