Chromatin-specific hypersensitive sites are assembled on a Xenopus histone gene injected into Xenopus oocytes.

A cloned histone H4 gene of Xenopus laevis is efficiently transcribed after injection into germinal vesicles of X. laevis oocytes. Deletion analyses indicate that less than 140 base-pairs of 5' flanking sequences and 50 base-pairs of 3' flanking sequences are required for efficient transcription of this gene in Xenopus oocytes. Chromatin footprint analysis by a direct end-label technique reveals discrete DNase I-hypersensitive and micrococcal nuclease-hypersensitive sites at the 5' and 3' boundaries of the gene, which bracket the transcribed region of this minichromosome. The specific chromatin structure assembled around this homologous gene, together with the finding that histone genes of Drosophila melanogaster are not assembled into specific nucleoprotein structures within Xenopus oocytes, strongly suggest that sequence-specific and species-specific factors may be responsible for generating the chromatin-specific hypersensitive sites at the boundaries of active genes.

A simple procedure for parallel sequence analysis of both strands of 5'-labeled DNA.

Ligation of a 5'-labeled DNA restriction fragment results in a circular DNA molecule carrying the two 32Ps at the reformed restriction site. Double digestions of the circular DNA with the original enzyme and a second restriction enzyme cleavage near the labeled site allows direct chemical sequencing of one 5'-labeled DNA strand. Similar double digestions, using an isoschizomer that cleaves differently at the 32P-labeled site, allows direct sequencing of the now 3'-labeled complementary DNA strand. It is possible to directly sequence both strands of cloned DNA inserts by using the above protocol and a multiple cloning site vector that provides the necessary restriction sites. The simultaneous and parallel visualization of both DNA strands eliminates sequence ambiguities. In addition, the labeled circular molecules are particularly useful for single-hit DNA cleavage studies and DNA footprint analysis. As an example, we show here an analysis of the micrococcal nuclease-induced breaks on the two strands of the somatic 5S RNA gene of Xenopus borealis, which suggests that the enzyme may recognize and cleave small AT-containing palindromes along the DNA helix.

The impact of new technologies on vaccines.

Vast changes are taking place in vaccinology consequent to the introduction of new technologies. Amongst the vaccines included in the Expanded Programme of Immunization (EPI), the pertussis vaccine has been replaced by acellular purified fractions devoid of side-effects. Non-pathogenic but immunogenic mutants of tetanus and diptheria toxins are likely to replace the toxoids. An effective vaccine against hepatitis B prepared by recombinant technology is in large-scale use. Conjugated vaccines against Haemophilus influenzae b, S. pneumococcus and meningococcus are now available, as also vaccines against mumps, rubella and measles. Combination vaccines have been devised to limit the number of injections. Vaccine delivery systems have been developed to deliver multiple doses of the vaccine at a single contact point. A genetically-engineered oral vaccine for typhoid imparts better and longer duration of immunity. Oral vaccines for cholera and other enteric infections are under clinical trials. The nose as a route for immunization is showing promise for mucosal immunity and for anti-inflammatory experimental vaccines against multiple sclerosis and insulin-dependent diabetes mellitus. The range of vaccines has expanded to include pathogens resident in the body such as Helicobacter pylori (duodenal ulcer), S. mutans (dental caries), and human papilloma virus (carcinoma of the cervix). An important progress is the recognition that DNA alone can constitute the vaccines, inducing both humoral and cell-mediated immune responses. A large number of DNA vaccines have been made and shown interesting results in experimental animals. Live recombinant vaccines against rabies and rinderpest have proven to be highly effective for controlling these infections in the field, and those for AIDS are under clinical trial. Potent adjuvants have added to the efficacy of the vaccines. New technologies have emerged to 'humanize' mouse monoclonals by genetic engineering and express these efficiently in plants. These recombinant antibodies are opening out an era of highly specific and safe therapeutic interventions. Human recombinant antibodies would be invaluable for treating patients with terminal tetanus and rabies. Antibodies are already in use for treatment of cancer, rheumatoid arthritis and allergies. An advantage of preformed antibodies directed at a defined target and given in adequate amounts is the certainty of efficacy in every recipient, in contrast to vaccines, where the quality and quantum of immune response varies from individual to individual.

Nylon friction dermatitis: A distinct subset of macular amyloidosis.

43 patients were taken up for the study, all of whom were asymptomatic and presented with bluish black pigmentation. 23 patients presented with pigmentation which was proximal and distal to the bony prominences, all of whom gave a history of using nylon scrubbers during bathing. 20 patients gave no history of friction and the pigmentation was present on the extensor forearms, shins and upper back. Histopathological examination confirmed amyloid deposits.

Hereditary motor and sensory neuropathy mimicking hansen's disease.

A rare case of hereditary motor and sensory neuropathy in a 45 year old man with glove and stocking hypoaesthesia, bilateral clawing, foot drop and thickening of the peripheral nerves mimicking Hansen's disease is reported.

Congenital multiple cutaneous mastocytoma.

A rare case of multiple cutaneous mastocytoma presenting at birth with multiple skin coloured to hyperpigmented papulonodules and plaques all over the body is being reported.

Primary cutaneous phaeohyphomycosis.

A rare case of phaeohyphomycosis presenting with a solitary nodule on right lower leg of 2 years duration is being reported. The disease showed marked response to oral fluconazole.

The efficacy of flutamide, an antiandrogen in idiopathic hirsutism.

The efficacy of flutamide, an antiandrogen in idiopathic hirsutism was studied. The long term effects of. treatment with low doses of flutamide on clinical and hormonal parameters were investigated. Nine patients with idiopathic hirsutism were studied basally and during treatment with 125mg flutamide thrice daily for a period of 9 months. Safety parameters were assessed throughout the study. Hirsutism was graded by Ferriman and Gallwey score and hormones were evaluated basally and later quarterly. After three months of therapy, flutamide had caused a significant alleviation of hirsutism and this continued during the subsequent months. No clinical significant side effects were observed during the period of the study. Biochemical and hormonal parameters remained unchanged after 9 months of flutamide.

Assembly of transcriptionally active chromatin in Xenopus oocytes requires specific DNA binding factors.

Active minichromosomes assembled on injected 5S RNA gene clones are stable in Xenopus oocytes; endogenous 5S DNA specific factor(s) are required for their assembly. When somatic-type and oocyte-type 5S RNA gene clones are coinjected, the somatic genes are assembled into active minichromosomes, while most of the oocyte genes are assembled into inactive ones. The differential 5S RNA gene expression, which mimics that in somatic cells, appears to result from titration of 5S DNA specific factor(s) by the competing somatic 5S DNA, followed by histone mediated assembly of inactive chromatin on the oocyte 5S DNA. Stable minichromosomes are also assembled on a cloned histone H4 gene; again, intragenic DNA rearrangements affect the efficiency of assembly of active chromatin and differential gene expression occurs after coinjection of two or more H4 DNA constructs. We suggest that the H4 DNA molecules also compete for limiting quantities of specific DNA binding factor(s) required for the assembly of active H4 gene chromatin.

The role of DNA-mediated transfer of TFIIIA in the concerted gyration and differential activation of the Xenopus 5S RNA genes.

The transcription factor of the 5S RNA gene, TFIIIA, induces gyration of oocyte- and somatic-type 5S DNA plasmids in Xenopus oocyte extracts, but oocyte 5S gyration requires a 5-fold higher TFIIIA concentration than somatic 5S gyration. Concomitant with the differential gyration at intermediate TFIIIA concentrations, the oocyte genes are repressed and the somatic genes become activated, a situation that mimics the one seen in Xenopus somatic cells. Data obtained with plasmids immobilized in agarose indicate that TFIIIA finds its site via a DNA-mediated transfer mechanism, and that all-or-none gyration is a consequence of TFIIIA transfer between 5S DNA sites. Based on these results, we present a model that explains the differential all-or-none activation of the 5S RNA genes.

Analogous cleavage of DNA by micrococcal nuclease and a 1-10-phenanthroline-cuprous complex.

We have examined the DNA cleavage specificity of a 1,10-phenanthroline-cuprous complex and find this agent to recognize the same sites and cleave with the same relative preferences as micrococcal nuclease. In contrast, DNase I and bleomycin-ferrous complex cleave the same 5000 bp D. melanogaster histone gene DNA with different specificities, although some of the sites appear to be recognized and cleaved by all four reagents. Our results suggest that the reagents used probably detect discrete conformational perturbations along the DNA double helix.

Accessory and alloantigen-presenting cell functions of A431 keratinocytes that stably express the B7 antigen.

Because keratinocytes are tolerogenic antigen-presenting cells (APC), we investigated the role of B7/BB-1 in reconstituting defective accessory cell (AC) and APC functions by such nonlymphoid cells. KC were induced to stably express B7/BB-1 by DNA-mediated gene transfection. This single-transformed B7/BB-1+ A431 cell line (but not control A431) functioned as AC for lectin-induced T-cell proliferation, as well as oxidative mitogenesis. This costimulation was dependent on B7/BB-1 expression since the monoclonal antibody BB-1 blocked costimulation of PHA mitogenesis by 75%. After the induction of class II MHC antigen expression by interferon-gamma, B7/BB-1+ KC but not control KC presented alloantigens to resting T-cells in the primary mixed lymphocyte reaction. These data indicate that the stable expression of B7/BB-1 antigen by KC reconstitutes defective AC and alloantigen-presenting activity. The lack of expression of such second signal by normal KC may be responsible for their ability to induce clonal anergy in vitro and in vivo.

Use of oral fluorescein angiography in the diagnosis of macular oedema within a diabetic retinopathy screening programme.

AIMS: To assess if oral fluorescein angiography (OFA) is a suitable screening method to detect macular oedema in diabetic retinopathy. METHODS: Eighty-four diabetic patients were included in the study. They were from a consecutive series of patients attending the diabetic eye-screening clinic, with retinopathy at the macula requiring ophthalmology assessment. All patients were subsequently examined in the eye hospital, by ophthalmologist slit lamp biomicroscopy assessment as the gold standard, followed by oral fluorescein angiography. RESULTS: This study indicates a sensitivity of 92% and specificity of 81%. Only 4.8% of patients developed a minor reaction to oral fluorescein; 84.5% of images were of good quality. CONCLUSIONS: Oral fluorescein angiography is an efficient and highly sensitive tool for the detection of macular oedema. It can be used as an adjunct in the diabetic screening service to identify patients with oedema within a disc diameter of the macula. Ultimately it will ensure that only necessary and smaller numbers of patients are referred to ophthalmologists.

Organization and evolution of the human epidermal keratinocyte transglutaminase I gene.

Transglutaminases (TGases; protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13) are calcium-dependent crosslinking enzymes that modify proteins posttranslationally. Several distinct types of TGases have been identified, which appear to be encoded by a family of closely related genes. We isolated the gene encoding human keratinocyte-specific type I TGase (TGase I) and characterized its chromosomal organization. The TGase I gene consists of 15 exons separated by 14 introns and exhibits a restriction fragment length polymorphism. Exons appear to encode functional and/or structural domains: exon I and part of exon XV encode untranslated regions, whereas exons VII and XI contain the active site and a presumptive calcium-binding domain, respectively. Interestingly, exon VI of TGase I contains a consensus Arg-Gly-Asp tripeptide sequence whose presence suggests an intriguing extracellular function for the enzyme. We present a likely phylogenetic tree for seven known members of the TGase family based on amino acid sequence similarity. Arguments presented suggest that the active enzyme evolved first and the structural human erythrocyte membrane protein 4.2 (band 4.2) has undergone a rapid change in amino acid sequence. It follows that band 4.2 evolved from the type II TGases, whereas factor XIII subunit a evolved from the type I group.