Cleavage of Müllerian inhibiting substance activates antiproliferative effects in vivo

Müllerian inhibiting substance (MIS), an inhibitor of growth and development of the female reproductive ducts in male fetuses, requires precise proteolytic cleavage to yield its biologically active species. Human plasmin is now used to cleave and, thereby, activate immunoaffinity-purified recombinant human MIS at its monobasic arginine-serine site at residues 427-428. To avoid the need for exogenous enzymatic cleavage and to simplify purification, we created an arginine-arginine dibasic cleavage site (MIS RR) using site-directed mutagenesis to change the serine at position 428 (AGC) to an arginine (cGC). The mutant cDNA was then stably transfected into a MIS-responsive ocular melanoma cell line, OM431, followed by cloning for amplified expression to test its biological activity in vitro and in vivo. Media from each clone were assayed for production of MIS RR by a sensitive ELISA for holo-MIS, and high- and low-producing clones were selected for further study. Media from the highest MIS RR producer caused Müllerian duct regression in an organ culture bioassay. Other transfections were done with an empty vector (pcDNAI Neo) or a construct lacking the leader sequence and thus failing to secrete MIS, to serve as controls. The OM431 clones containing the MIS RR mutant were growth inhibited in monolayer culture. The high- and low-producing MIS RR OM431 clones, along with transfected OM431 controls, were injected into the tail veins of immunosuppressed severe combined immunodeficiency mice for in vivo analyses. Four to 6 weeks later, pulmonary metastases were counted in uniformly inflated lungs. OM431 clones containing the more easily cleaved MIS RR displayed a significant dose-dependent reduction in pulmonary metastases when compared to the lungs of animals given injections of OM431 clones containing empty vector, leaderless MIS, or wild-type MIS that requires activation by plasmin cleavage. Since the purification protocol of MIS RR is less complicated than that for wild-type MIS, which requires subsequent enzymatic activation, MIS RR can be used for scale-up production with increased yields for further therapeutic trials against MIS-sensitive tumors.     

The synthesis of symmetrical and unsymmetrical P1/P1' cyclic ureas as HIV protease inhibitors

Cyclic urea SD146, a potent HIV protease inhibitor bearing a flat resistance profile, possessed poor solubility and bioavailability, which precluded further development of the compound. In an effort to improve upon the pharmacokinetic profile of the compound, several analogs modified at the P1/P1' residues were prepared and evaluated. Several of those compounds displayed significant improvement of physical properties.     

Current options in prosthetic vascular graft infection

Debate continues over which procedure is the best treatment for prosthetic graft infections. We retrospectively reviewed the medical records at our institution for all vascular graft infections that occurred from 1985 to 1995 to evaluate their occurrence, treatment, and outcome. Twenty-four patients had prosthetic graft infections. The average patient age was 62 years, and 67 per cent of the patients studied were men. The initial operation was for treatment of occlusive disease in 92 per cent of the patients, and aortofemoral bypasses were the most common procedures performed (15 of 24 patients, 63%). The average interval from graft implantation to presentation of infection was 29 months. In lower-extremity bypasses, the site of infection was most commonly in the groin (87%). Gram-positive organisms, including coagulase-negative Staphylococcus (32%) and Staphylococcus aureus (28%), were the most frequently isolated bacteria. Thirty procedures were performed for management of the graft infections. Extra-anatomic bypass was associated with no recurrent graft infections. Graft preservation was successful in two cases of early S. aureus infection (less than 1 year after original procedure), and in situ graft replacement was successful in all four cases of late-appearing coagulase-negative Staphylococcus infection (more than 1 year after original procedure). Both treatments failed in all five cases of Gram-negative infection (P = 0.008 by Fisher's exact test). The overall mortality and amputation rates were 17 per cent and 21 per cent, respectively, without significant differences between the treatment modalities. Extra-anatomic bypass remains the best treatment for prosthetic graft infection. In situ replacement and graft preservation treatments should be selective and based on presentation of the infection and the type of pathogenic organism.     

Developmental stages in experimental liver metastases: relation to invasiveness

We have previously reported that an invasive morphotype can be evoked in a rat colon carcinoma by transplanting it into pre-induced subcutaneous granulation tissue. We have now studied the interaction of the same tumor with liver tissue, which is extremely poor in connective tissue in comparison with the subcutaneous site. Tumor cells were injected into the portal system and the resulting experimental liver metastases were examined by electron microscopy and immunohistochemistry. Early metastases consisted of well-differentiated acini, fully surrounded by connective tissue that was derived from the periportal stroma. In a later stage, this connective tissue was overgrown by tumor cells and, almost immediately, acinar differentiation was lost. Most metastases eventually reached the liver capsule, which reacted by forming a layer of granulation tissue. Only in this layer, we observed invasion by thin tumor cell strands, which were often intimately associated with fibroblasts or with blood capillaries. The tumor cells remained smooth and rounded during this process. After fully penetrating the granulation tissue, the tumor cell strands reached the liver surface, where they formed poorly structured papillary masses that were nearly devoid of stroma. Our observations indicate that, even in a relatively homogeneous organ like the liver, the tumor-host interaction is highly complex and dynamic. They also confirm the notion that granulation tissue stimulates tumor invasiveness. Finally, they show that tumor cells can actively invade host tissues without exhibiting a "fibroblastic" morphology.     

Lichtenstein herniotomy

All standard methods of hernia repair involve suturing together tissues which are not normally in apposition. This violates the basic surgical principle that tissue must never be approximated under tension and accounts for an unacceptable number of failures. Total reinforcement of the inguinal floor with a sheet of suitable biomaterial and employment of a "tension-free" technique is a more effective approach. Since June 1984, 3250 primary inguinal hernias have been repaired at the Lichtenstein Hernia Institute by the open tension-free technique using Marlex mesh. All operations were performed under local anesthesia. Patients were discharged from the hospital within two or four hours after the operation. The patients were followed from one to 8 years by physician examination. The follow-up rate was 87%. There were four recurrences. The causes of recurrence and how to avoid them are discussed.     

Primary structure of gene H of cattle plague virus strain K]

The paper analyzes the standard legal and methodological assurance of the quality and safety of animal food raw materials and foodstuffs (meat, meat products, fish, shellfish, crayfish and their processing products) by the parasitic purity rates according the requirements under the Russian Federation's laws "On Sanitary and Epidemiological Well-Being of the Population", "On Protection of Consumer's Rights", "On Certification of Products and Services", those of SanPiN, such as 2.3.2.560-96 "Sanitary Requirements for the Quality and Safety of Food Raw Materials and Foodstuffs" and 3.2.569-96 "Prevention of Parasitic Diseases in the Russian Federation".     

Detection of serum antibodies to Chlamydia trachomatis in patients with chlamydial and nonchlamydial pelvic inflammatory disease by the IPAzyme Chlamydia and enzyme immunoassay

A novel serological test, IPAzyme Chlamydia (Savyon Diagnostics Ltd., Beer Sheva, Israel), was compared with an enzyme immunoassay (EIA) for the ability to detect serum immunoglobulin G and A antibodies in the diagnosis of acute chlamydial pelvic inflammatory disease. In comparison with cell culture, which is the "gold standard," IPAzyme Chlamydia and EIA exhibited sensitivities of 63 and 68% and specificities of 76 and 87%, respectively. Thus, IPAzyme Chlamydia offers no advantages over the EIA, and neither serological test can be recommended for the diagnosis of acute Chlamydia trachomatis infection. So far, conventional cell culture remains the most reliable diagnostic test for chlamydial pelvic inflammatory disease.     

Ultrastructure of nerve plexus in flatworms. III. The infra-epithelial nervous system

In addition to a submuscular and subepithelial nerve plexus an infra-epithelial nerve-net also occurs in Notoplana acticola. It contains naked nerve cells which lie between the bases of epithelial cells. Individual neurites occur separately or in small tracts. Synapses contain clear spherical vesicles and are polarized. Post-synaptic neurites tend to be flat and ribbon-like. Rhabdite and mucus secreting cells in the epithelium have synapses associated with them. Neurites ending on the rhabdites appear to originate in the subepithelial plexus. The "sensory-free nerve endings" of earlier workers are reinterpreted as being motor terminals on epithelial secretory cells.