Selective chemical modification of cytochrome P450scc (CYP11A1) with diiodofluorescein iodoacetamide in the study of the role and topology of interdomain hinge of the hemoprotein molecule

Bovine adrenocortical cytochrome P450scc (CYP11A1) was selectively modified with diiodofluorescein iodoacetamide (DIFIA). Only Cys264 is labeled in the P450 polypeptide chain. The modification significantly affected the cholesterol-hydroxylating activity in the reconstituted system containing NADPH, adrenodoxin reductase, adrenodoxin, and soluble or membrane-bound P450scc. The inhibitory effect correlates with decreased affinity of cytochrome P450scc to intermediate electron carrier, adrenodoxin. Cytochrome P450scc is modified in liposomes and the modified membrane-bound protein is cleaved by trypsin forming two large fragments F1 and F2 corresponding to the N- and C-terminal regions of the molecule. The data indicate that the Cys264-containing region of the cytochrome P450scc molecule is exposed to the surface of protein globule, located outside of the membrane, and can participate in protein-protein interactions.     

Anti-inflammatory activity of some novel 1-[3-(aroylo)] and one 1-[3-(aryloxy)]-propyl aminothiazole in correlation with structure and lipophilicity

The effect of some new 1-[3-(aroylo)] and one 1-[3-[aryloxy)]-propyl aminothiazole on inflammation was investigated in the carrageenin-induced mouse paw edema (36-58% edema inhibition). In addition they exhibited significant analgesic activity (55-80.6%) in the writhing test. Their antioxidant activity in vitro using the stable free radical 1.1-diphenyl-2-picrylhydrazyl (DPPH), was determined. Antiproteolytic ability and beta-glucuronidase inhibition have been studied. The tested compounds did not inhibit soybean 12-lipoxygenase. RM values, as an expression of lipophilicity, were determined.     

Principles of biochemical diagnosis of early infection process

The paper outlines major achievements of biochemical diagnosis of a wound infectious process. Various classifications of its methods are given. The most promising approaches to predictive diagnosis of wound suppuration, pyoresorptive fever and sepsis are discussed.     

The pericentromeric inversion, inv (6)(p25q13), is a novel diagnostic marker in chondromyxoid fibroma

Chondromyxoid fibroma is a benign bone tumor that arises most commonly in the metaphysis of long bones in young adults. The cytogenetic features of this tumor are not well known. In this study, four chondromyxoid fibromas were karyotyped after short-term cell culture. All of the tumors contained clonal rearrangements of chromosome 6, and each of these rearrangements involved band 6q13. Two tumors contained a pericentromeric inversion, inv (6)(p25q13), which was described recently in chondromyxoid fibroma. 6q13 rearrangements are not associated with other types of bone and soft tissue tumors, and the inv (6)(p25q13) is reported only in chondromyxoid fibroma. Hence, this cytogenetic marker might be helpful in distinguishing chondromyxoid fibroma from chondrosarcoma and other histologic mimics. The consistent occurrence of 6q13 rearrangements suggests a specific oncogenic mechanism in chondromyxoid fibroma, most likely involving oncogene activation.     

An investigation into the role of waterproofing in a two-stage hypospadias repair

In our unit a two-stage procedure, using a full thickness preputial graft, has been adopted as the method of choice for the repair of hypospadias proximal to the coronal sulcus. In 1993 an audit was undertaken to establish our complication rate for this procedure. Twenty-two consecutive patients who completed a two-stage repair between January 1988 and December 1993 were studied. An unacceptably high fistula rate was identified (63%, 14/22 cases). Consequently our technique was modified by transposing a vascularised flap of preputial areolar tissue over the urethral suture line, at the time of urethroplasty. A second group of 22 consecutive patients, operated upon between January 1994 and July 1997, were subsequently investigated and a dramatic improvement in the fistula rate was demonstrated (4.5%, 1/22 cases). These cases represent a subgroup of almost 200 cases, which the senior author has managed over the last 10 years. The senior author undertook or supervised the surgery in all 44 cases, which were the focus of this study, and the introduction of a waterproofing layer represents the only change in technique.     

CTLA-4 blockade synergizes with tumor-derived granulocyte-macrophage colony-stimulating factor for treatment of an experimental mammary carcinoma

Generation of a T cell-mediated antitumor response depends on T cell receptor engagement by major histocompatibility complex/antigen as well as CD28 ligation by B7. CTLA-4 is a second B7 receptor expressed by T cells upon activation that, unlike CD28, appears to deliver an inhibitory signal to T cells. Recently, we and others demonstrated that administration of an anti-CTLA-4 antibody was sufficient to promote regression of several murine tumors. However, certain tumors, such as the SM1 mammary carcinoma, remain refractory to this type of immunotherapy. In the present study, we report that the combination of both CTLA-4 blockade and a vaccine consisting of granulocyte-macrophage colony-stimulating factor-expressing SM1 cells resulted in regression of parental SM1 tumors, despite the ineffectiveness of either treatment alone. This synergistic therapy resulted in long-lasting immunity to SM1 and depended on both CD4(+) and CD8(+) T cells. Interestingly, synergy was not observed between CTLA-4 and a B7-expressing SM1 vaccine. Given that granulocyte-macrophage colony-stimulating factor promotes differentiation and activation of dendritic cells as well as enhances cross-priming of T cells to tumor-derived antigens and that SM1 is major histocompatibility complex class II-negative, our findings suggest that CTLA-4 blockade acts at the level of a host-derived antigen-presenting cell. In addition, these results also support the idea that the most effective and synergistic vaccine strategy targets treatments that enhance T cell priming at the level of host-derived antigen-presenting cells.     

Metastatic vertebral disease in children

We retrospectively reviewed 19 children with osseous vertebral metastases. The average age was 10.9 years at diagnosis of the primary tumor and 11.2 years at diagnosis of metastasis. The location of the multiple metastases was cervical (three), thoracic (14), and lumbar (10). Treatment consisted of chemotherapy (19), radiation therapy (12), and operation (seven). Ten children died at an average of 13 months after diagnosis of the primary tumor; nine are alive at an average of 72 months after diagnosis. All four children treated with chemotherapy, spinal radiation, and laminectomy, and who survived > 2 months, developed deformity. In this particular situation, we encourage consideration of spinal stabilization when it is appropriately coordinated with radiation and chemotherapy.     

Radiosensitivity of atomic bomb survivors as determined with a micronucleus assay

We have developed a system in which a unique double-stranded break (DSB) can be introduced into a yeast chromosome during mitotic growth. The recognition site for the endonuclease I-SceI was inserted at different places in the yeast genome in haploid and diploid cells expressing this endonuclease. Induction of the break in haploids results in cell death if no intact copy of the cleaved region is present in the cell. If such a copy is provided on a plasmid, as an ectopic gene duplication, or on a homologous chromosome, the break can be repaired. Repair results in two identical copies in the genome of the locus which has been cut. We call this phenomenon homozygotization by reference to diploids heterozygous for the cut site in which repair leads to homozygosis at this site. We have compared the efficiencies of repair in the various topological situations examined, and conclude that some mechanism must search for regions of homology to both sides of the DSB and that repair is successful only if the homologies are provided by the same template molecule.