Cell membrane-associated MT1-MMP-dependent activation of pro-MMP-2 in A375 melanoma cells.

Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases, can degrade extracellular matrix components under physiological conditions and during cancer invasion and metastasis. Among the MMPs, the 72 kDa type IV collagenase MMP-2 (gelatinase A) is activated in a membrane-associated manner by an activation complex composed of membrane type 1 matrix metalloproteinase (MT1-MMP), tissue inhibitor of matrixmetalloproteinase-2 (TIMP-2), and pro-MMP-2 in the presence of alphavbeta3 integrin receptor. The activation of pro-MMP-2 correlates with increased occurrence of metastases. Increased MMP-2 activity has been demonstrated in many human tumors. In the present communication, we studied cell surface-associated activation of MMP-2 (72 kDa collagenase type IV) in the moderately metastatic human melanoma cell line A375. RESULTS: Activation of purified 72 kDa collagenase type IV, pro-MMP-2 from cervical cancer tissue homogenate and from serum-free culture medium of HT1080 cells grown in presence of concanavalin A, by A375 cells, was shown by gelatin zymography. A375 cells activated only pro-MMP-2 from purified MMP-9/MMP-2 mixture indicating that the activation is specific for MMP-2. Activation of MMP-2 and purified collagenase type IV by A375 membrane fraction and membrane extract was also demonstrated by gelatin zymography. When A375 cells were first incubated with anti-MT1-MMP polyclonal antibody, activation of collagenase type IV was significantly decreased, indicating that membrane-associated MMP-2 activation is MT1-MMP-mediated. Immunocytochemistry showed MT1-MMP localization at focal adhesion sites. The presence of the components of activation complex-MT1-MMP and integrin alphavbeta3-were confirmed by Western blot, cell adhesion assay, and integrin subunit assay. CONCLUSION: Our experimental findings furnish another example of the unique membrane-associated MMP-2 activation mechanism in A375 melanoma cells and clearly indicate the role of MT1-MMP in MMP-2 activation. The information could help in developing new therapies designed to interfere with MMP activation and management of cancer and metastases.     

Extracorporeal membrane oxygenation with lepirudin anticoagulation for Wegener's granulomatosis with heparin-induced thrombocytopenia

Venovenous extracorporeal membrane oxygenation with lepirudin anticoagulation was successfully used for a complicated case of Wegener's granulomatosis and heparin-induced thrombocytopenia. Interestingly, a linear correlation was found between activated partial thromboplastin time and activated clotting time during lepirudin anticoagulation.     

Comparison of capsid polypeptides of group B coxsackieviruses and polypeptide synthesis in infected cells

Summary Capsid polypeptides of all six types (B1–6) of group B coxsackieviruses were compared by high-resolution gel electrophoresis, and synthesis of protein and RNA in B4- or B5-infected HeLa cells was analyzed. Four polypeptides, VP1–4, were detected in each type. Another polypeptide, VP0, slightly larger than VP1, was also detected in trace amounts in some types. VP1–3 showed different but characteristic molecular weights (VP1, 34,500 to 37,000; VP2, 31,000 to 36,000; VP3, 26,000 to 32,500), and presented well-defined and reproducible differences in electrophoretic mobility. The molecular weight of VP4 ranged from 5,000 to 5,500. VP1 was largest in B2 and B4, smallest in B1, and of intermediate size in the other types. VP2 was largest in B4 and smallest in B2; VP3 was largest in B5 and B6 and smallest in B4. In B4- or B5-infected HeLa cells, host protein synthesis began to decline after 2 hours postinfection and was less than 20 percent of the control by 6 hours postinfection. Actinomycin D-resistant viral RNA synthesis started at about 2 hours postinfection, peaked by 5 hours, and then declined rapidly. Virus-specific protein synthesis began while host protein synthesis was declining, increased during the ensuing period, and declined in late infection. A number of virus-specific proteins with molecular weights from 23,500 to >92,500 were detected in the host cytoplasm. At least three of these proteins were also present in the nucleus. The kinetics of processing of virus-specific proteins were examined by pulse-chase experiments in B5-infected cells. The relative intensities of [³⁵S]-methionine-labeled polypeptides suggest that a number of smaller, stable chains (MW 23,500 to 38,000) are generated by cleavage of a precursor polypeptide (MW 92,500 to 100,000).With 7 Figures     

Needle necropsy in AIDS

The value of autopsy in understanding the natural course of any disease is beyond any argument. The reluctance of pathologists to perform autopsy in HIV infected cadavers is justified due to the risks involved to the prosector and the morgue attendants. A relative low risk needle necropsy protocol is proposed using fine needle aspiration cytology, tru-cut biopsies and microbiological examination. Diagnosis could be offered in all the forty-four needle necropsies performed. Disseminated tuberculosis in 18/44 (40.9%) cases, disseminated cryptococcosis in 12/44 (27.2%) cases, poly-microbial infections in 27.2% cases and non-Hodgkin's lymphoma in 9% cases were detected in the study. Infectious agents like Histoplasma capsulatum, Isospora belli, tachyzoites of Toxoplasma gondii, Candida sp and Cryptococcus sp could be demonstrated in the samples obtained in the study. Lack of material for study of gross pathology, inaccessibility of deep-seated lesions and risk of needle stick injury to the prosector though low are the limitations of this procedure.     

Identification of a new genotype H wild-type mumps virus strain and its molecular relatedness to other virulent and attenuated strains

A single clinical isolate of mumps virus designated 88-1961 was obtained from a patient hospitalized with a clinical history of upper respiratory tract infection, parotitis, severe headache, fever and lymphadenopathy. We have sequenced the full-length genome of 88-1961 and compared it against all available full-length sequences of mumps virus. Based upon its nucleotide sequence of the SH gene 88-1961 was identified as a genotype H mumps strain. The overall extent of nucleotide and amino acid differences between each individual gene and protein of 88-1961 and the full-length mumps samples showed that the missense to silent ratios were unevenly distributed. Upon evaluation of the consensus sequence of 88-1961, four positions were found to be clearly heterogeneous at the nucleotide level (NP 315C/T, NP 318C/T, F 271A/C, and HN 855C/T). Sequence analysis revealed that the amino acid sequences for the NP, M, and the L protein were the most conserved, whereas the SH protein exhibited the highest variability among the compared mumps genotypes A, B, and G. No identifying molecular patterns in the non-coding (intergenic) or coding regions of 88-1961 were found when we compared it against relatively virulent (Urabe AM9 B, Glouc1/UK96, 87-1004 and 87-1005) and non-virulent mumps strains (Jeryl Lynn and all Urabe Am9 A substrains).     

Effect of the mode of croscarmellose sodium incorporation on tablet dissolution and friability

A computer-optimized experimental design was used to study the effect of incorporating a "super disintegrant", croscarmellose sodium, intragranularly, extragranularly, or distributed equally between the two phases of a tablet in which a poorly soluble drug constituted at least 92.5% of the formulation. The results were analyzed by means of a general quadratic response surface model and suggest that tablets with the same total concentration of super disintegrant dissolve at a faster rate when the super disintegrant is included intragranularly. Tablet friability was not affected by the method of super disintegrant incorporation.     

Rapid diagnosis of early ectopic pregnancy in an emergency gynaecology service--are measurements of progesterone, intact and free {beta} human chorionic gonadotrophin helpful?

The clinical usefulness of measuring serum concentrations ofprogesterone, human chorionic gonadotrophin (HCG) and the free-subunit of HCG in distinguishing between early viable and non-viablepregnancy, before an accurate ultrasound diagnosis is possible,was evaluated in a prospective study of patients presentingto our emergency gynaecology service with a clinical suspicionof ectopic pregnancy. Patients were selected on the basis ofinitial HCG concentrations; samples with HCG 25–10 000IU/I were later analysed for progesterone and free HCG. Of the181 patients studied, 38 (21%) had an ectopic pregnancy, 108(60%) had a spontaneous abortion and 35 (19%) had a viable intra-uterinepregnancy. Concentrations of HCG and free HCG in the group withviable pregnancies were significantly higher than in the groupwith ectopic pregnancy (P < 0.001) and than those destinedto miscarry (P < 0.01). Progesterone concentrations werealso significantly higher in the viable versus the ectopic andthe spontaneous abortion groups (P < 0.001 in each case).Despite these highly significant differences there was a degreeof overlap such that it was impossible to devise a cut-off levelfor any hormone analysed, either singly or in combination, whichwould offer a clinically useful predictor of outcome.     

Communicating health

Routine production of communication materials without paying attention to utilization, field test, and impact analysis is ineffective. The concept of information, education, and communication (IEC) should encompass voluntary activity of health education in a tradition of innovation. One seminal factor may be the communication technologies developed by the National Technology Missions. The missions were participatory by seeking solutions among communities and analyzing health issues from the perspective of those directly involved, rather than from the top down. The prime focus of the national drinking water mission was convenience, hence messages concentrating on health advantages were ignored. At this juncture, influencing health behavior required decentralization reflecting local cultures. Thus community-based partners became the foundation of a strategy of communicating safe water. As national strategies emerged in each of the technology missions, communication addressed advocacy of the need for political will, dissemination of technical information, and influencing patterns of behavior. Despite learning a new understanding, the danger exists that IEC remains just another label of mass communication with posters, advertisements, brochures, radio, and television. Decisions on contraceptive choice and use requires more than just accurate information; it requires the power to make such a decision. A new approach demands a priority for communication skills taking into account people's aspirations. The HIV-AIDS crisis underlines the urgency with which communication has to respond to health challenges. A series of experiments facilitated by the World Conservation Union helped build communication capabilities among environmental groups working in Latin America, Africa, and India. The International Reference Center on Water and Sanitation initiated pilot communication projects in West Africa for community health.