The role of insulin-like growth factor-1 (IGF-1) and IGF binding protein-1 (IGFBP-1) in the pathogenesis of polycystic ovary syndrome. The objective of this study was to elucidate the relationship and role of insulin-like growth factor-1 (IGF-1), IGF binding protein-1 (IGFBP-1), insulin and luteinizing hormone (LH) in the pathogenesis of polycystic ovary syndrome (PCOS). In a pilot study, serum concentrations of IGF-1 were determined in women with PCOS (n = 10), hypopituitarism (n = 12) and normal controls (n = 10). In the main study, serum concentrations of IGF-1, IGFBP-1, insulin and LH in women with anovulation associated (n = 23) and not associated (n = 47) with PCOS were determined. Serum concentrations of IGF-1 were not different in women with PCOS, anovulatory non-PCOS and healthy women but were low in those with hypopituitarism. Mean serum IGFBP-1 in PCOS (33.8 +/- 21.2 micrograms/l) was decreased compared with anovulatory non-PCOS (60.0 +/- 22 micrograms/l) (P = 0.0001), and correlated negatively with insulin concentrations (r = -0.67, P = 0.0006). Patients with PCOS could be separated into those with high LH and those with high insulin levels. It was concluded that women with PCOS have normal serum IGF-1 concentrations but IGFBP-1 levels, regulated by insulin, are low. Hyperinsulinaemia and raised LH are independently capable of stimulating ovarian androgen production. Growth factors may have an important role in the pathogenesis of PCOS.


Recurrence of hyperthyroidism in multinodular goiter after long-term drug therapy: a comparison with Graves' disease. The chance of permanent remission after prolonged drug therapy was investigated in 41 patients with toxic multinodular goiter. For purposes of comparison a group of 41 patients with Graves' disease was also studied. After euthyroidism was achieved all patients received a combination of thionamide and thyroxine for at least 12 months. The minimum follow-up period was 2 yr. Relapse of thyrotoxicosis occurred in 95.1% of patients with toxic multinodular goiter and 34.1% of patients with Graves' disease (p < 0.001). It is concluded that for patients with toxic multinodular goiter early radioiodine therapy or surgery is preferred since prolonged drug therapy seldom produces permanent remission.


Antithyroid drug and Graves' hyperthyroidism. Significance of treatment duration and TRAb determination on lasting remission. To investigate the significance of treatment with antithyroid drugs longer than 12 months on lasting remission in Graves' hyperthyroid patients, and to study clinical and laboratory parameters of prognostic value. Fifty-two untreated Graves' hyperthyroid patients were assigned at random to two therapeutic groups. They were treated with carbimazole during 12 and 24 months in Group I (n = 28) and Group II (n = 24), respectively. Serum levels of FT4, T3, sTSH and TSH receptor antibody (TRAb) were measured before starting treatment and at regular intervals during treatment and follow-up after drug withdrawal. We compared the relapse rate in both groups of patients, at short (2-yr) and long-term (5-yr) periods after drug withdrawal. Also, we compared clinical and biochemical parameters between patients who stayed in remission and who had relapse. At the end of the short-term period, relapse had occurred in 13 (46.4%) Group I patients and in 13 (54.1%) Group II patients, p = 0.36. At the end of the long-term period, relapse had occurred in 24 (85.7%) Group I and 20 (83.3%) Group II patients, p = 0.78. No difference could be observed between patients who had stayed in remission and who had suffered relapse, within the 5-yr follow-up period regarding to goiter size, frequency of ophthalmopathy, TSH and TRAb levels. The high relapse rate observed could be due to high iodine intake in our country. In this study and in a review of the available data, we have been unable to find any rational basis for courses of antithyroid drugs longer than twelve months for the treatment of Graves' hyperthyroidism.(ABSTRACT TRUNCATED AT 250 WORDS)


A calcium conducting channel akin to a calcium pump. Calcium conducting channels were studied in blebs of sarcoplasmic reticulum described by Stein & Palade (1988). The calcium channels had at least three conductance states (70 pS, 50 pS and 37 pS) and were weakly selective for calcium ions, with a permeability ratio Ca2+ to K+ of about 3.4. The open probability of the channel was strongly voltage dependent, decreasing at positive membrane voltages. 10 microM ryanodine and 5 microM ruthenium red had no effect on this channel; neither did millimolar concentrations of ATP, Mg2+, caffeine, and Ca2+, implying that the calcium conducting channels are not ryanodine receptors. Several calcium pump inhibitors--namely, vanadate, AlF4-, reactive red 120, and cyclopiazonic acid--had obvious effects on the calcium conducting channels, suggesting that the calcium conducting channel of SR membrane blebs is some form of the SR calcium pump.


Calcium-independent K(+)-selective channel from chromaffin granule membranes. Intact adrenal chromaffin granules and purified granule membrane ghosts were allowed to fuse with acidic phospholipid planar bilayer membranes in the presence of Ca2+ (1 mM). From both preparations, we were able to detect a large conductance potassium channel (ca. 160 pS in symmetrical 400 mM K+), which was highly selective for K+ over Na+ (PK/PNa = 11) as estimated from the reversal potential of the channel current. Channel activity was unaffected by charybdotoxin, a blocker of the [Ca2+]-activated K+ channel of large conductance. Furthermore, this channel proved quite different from the previously described channels from other types of secretory vesicle preparations, not only in its selectivity and conductance, but also in its insensitivity to both calcium and potential across the bilayer. We conclude that the chromaffin granule membrane contains a K(+)-selective channel with large conductance. We suggest that the role of this channel may include ion movement during granule assembly or recycling, and do not rule out events leading to exocytosis.


Immunohistochemical study of the histogenesis of esophageal carcinosarcoma. In order to clarify the histogenesis and the direction of differentiation of spindle-cell and sarcomatous components of esophageal carcinosarcoma, 20 cases of the disease were reviewed histologically and immunohistochemically using the avidin-biotin-peroxidase complex method with monoclonal and polyclonal antibodies to various keratins, vimentin, desmin, muscle specific actin and S-100 protein. A gradual transition between carcinomatous and spindle cell sarcomatous components was present histologically in all 20 cases. Positive immunoreactivity for keratins was found in carcinomatous areas in all cases. Spindle cells in the transitional areas were positive for keratins in nine cases and for vimentin in five. Two cases demonstrated trace positive reactions to both keratin and vimentin in the same areas of transitional spindle cells between carcinomatous and sarcomatous components. The sarcomatous component showed an immunohistochemically positive reaction for vimentin in ten cases and for desmin in two. In one of the 20 cases, chondrosarcomatous cells were seen which showed a positive reaction to S-100 protein but were negative to keratin. The findings strongly suggested that neoplastic epithelial cells may show dedifferentiation to transforming spindle cells and also disdifferentiation to non-epithelial sarcoma like chondrosarcoma and leiomyosarcoma.


Immunohistochemical detection of Pneumocystis carinii in transbronchial lung biopsy specimens: antigen difference between human and rat Pneumocystis carinii. Transbronchial lung biopsy specimens, from three patients with non-AIDS-related Pneumocystis carinii pneumonia (PCP) and rat lung tissue in which PCP was induced by the administration of dexamethasone, were studied to determine the diagnostic usefulness of an immunohistochemical method using commercially available anti-Pneumocystis monoclonal antibody, 3F6, on formalin-fixed, paraffin-embedded tissue. PC was consistently stained a bright red color and unambiguously identified in all three human lung specimens, but not stained in lung tissues at autopsy from patients with various fungal pneumonias. In contrast, PC was weakly stained in PCP-induced rat lungs. The present study indicates human PC and rat PC to be antigenically different in terms of the existence of the 82 kilo-dalton (kD) antigen against which 3F6 is directed.


Comparison of various assay systems for prostate-specific antigen standardization. To avoid confusion between serum prostate-specific antigen (PSA) values among various assay systems, clinical studies on the possibility of conversion among detection values were performed. The assay kits used for the PSA comparisons were MARKIT-F PA, MARKIT-M PA, EIKEN PA, PA test WAKO, Ball ELSA PSA, E-Test Tosoh II PA, PROS-CHECK PSA, DELFIA PSA and TANDEM-R PSA. Using each kit, the standards attached to each assay system were detected, and 142 sera samples from benign hypertrophies or prostate cancers were assayed for serum PSA values. By detecting the standards for each kit, slopes were obtained which were almost identical to those obtained from original assay system. The coefficients of correlation among the PSA detection systems, using patients' sera, were very high, and linear regression lines were also obtained. The results suggest that almost identical serum PSA values may be detected either by multiplying by a coefficient to bring it to the standard or using the conversion formula.


Malignant sex-cord stromal tumor of the testis: report of a case with special reference to its unusual intracytoplasmic substructures. A case of malignant sex-cord stromal tumor (gonadal stromal tumor) in the left testis of a 42-year-old man with no sign of hormonal abnormality is reported. Histologically, a large portion of the tumor was composed of solid nests of round cells with a moderate amount of eosinophilic cytoplasm, but some areas contained small spaces somewhat resembling Call-Exner bodies and/or the follicular structures of granulosa cell tumors. In general, however, the tumor cells did not show a clear-cut differentiation into either Sertoli cells or granulosa cells. The tumor, because of its high mitotic rate, lymphatic, venous and capsular invasion, and metastasis to the para-aortic lymph nodes, was considered to be malignant. We simply designated this tumor, therefore, as a malignant sex-cord stromal tumor. An ultrastructural study revealed unique intracytoplasmic bodies composed of multiple stacks of tubular structures which superficially mimicked annulate lamellae. No lipid droplets, Reinke's crystalloids or Charcot-Böttcher filaments were seen. The rarity of the tumor and the presence of unusual substructures prompted us to report the case.


T-cell hybridomas recognizing the envelope proteins of Semliki forest virus: their sensitivity to endo/lysosomal protease and the antigenicity. Eight T-cell hybridomas were established from the draining lymph node of C3H mice immunized with Semliki forest virus (SFV). Six of them showed specificity toward viral-structure protein E2, while the remaining two clones included one with specificity to an other structural protein E1 and the other with specificity to C. The production of IL-2 by the E2 protein-specific T-cell hybridomas in the presence of SFV was suppressed by treating the antigen-presenting cells (APC) with ammonium chloride raising pH of the acidic compartments. It was found also that treatment of APC with a thiol protease inhibitor, leupeptin or E64, resulted in a reduced response of some of the E2-specific T-cell hybridomas. The E2 protein of SFV proved to be resistant at pH 7.0, and sensitive at pH 5.0 to in vitro cathepsin B treatment. In contrast, the E1 and C proteins proved to be resistant to both pH values. These results indicate that the thiol protease, probably cathepsin B, works as one of the enzymes group involved in antigen processing.


[A new approach to the intraoperative diagnosis of cholangitis and to the choice of surgical procedure in operations on the bile ducts]. The authors have developed a prompt and informative method for assessment of the depth and reversibility of the morphofunctional changes in the common bile duct (CBD) wall with the use of staining by hematoxylin basic, fuchsin-picric acid. Four types of the morphologic changes in CBD wall, which characterize the different degree of reversibility, were established. Depending of presence of these types, the corresponding surgical tactics is recommended.


Effects of sodium butyrate on the expression of sodium channels by neuronal cell lines derived from the rat CNS. We have studied the effects of sodium butyrate on cell morphology and the expression of mRNAs encoding voltage-gated sodium channels in five neuronal cell lines, B35, B50, B65, B103 and B104, all derived from the rat CNS. The cells were grown in medium supplemented with 2.5 mM sodium-n-butyrate and examined daily by phase-contrast microscopy. Sodium butyrate caused slowing of cell division and the formation of longer and more highly branched cytoplasmic processes than were present in untreated cells. Expression of sodium channel mRNA was analysed by PCR with primers that allow the transcripts encoding the different types of sodium channel to be distinguished according to the lengths of the PCR products. The identity of the PCR products was confirmed by restriction enzyme digestion. Southern blotting and hybridization with internal radiolabelled probes. Prior to sodium butyrate treatment, expression of sodium channel mRNA was largely restricted to B50 and B104 cells: B50 cells showed expression of rat brain types I and II sodium channel and B104 cells expressed rat brain type III sodium channel. After treatment for 5 days with sodium butyrate, sodium channel mRNA was detected in all five cell lines. In addition to type I and type II sodium channel, B50 cells expressed rat brain type III sodium channel. These three types of sodium channel were also expressed by B35, B65 and B103 cells. Even after butyrate treatment, B104 cells expressed only type III sodium channel. The treatment also induced expression of rat skeletal muscle SkM1 sodium channel in B35 cells but only trace amounts in the other neuronal cell lines.


The lipooligosaccharide immunotype as a virulence determinant in Neisseria meningitidis. We have studied the antigenic (immunotype) and physical characteristics of the lipooligosaccharide (LOS) of epidemiologically related Neisseria meningitidis case (36) and carrier (76) isolates associated with a virulent clone of meningococci (ET-5 complex). LOS immunotypes were determined by dot blotting using immunotype specific monoclonal antibodies and physical characteristics were determined from silver stained SDS-PAGE following proteinase K digestion. The genetic similarity of the different isolates was confirmed by analysis of the restriction fragment length polymorphisms. An association between LOS immunotype expression and invasive disease was found; 97% of case isolates expressed the L3,7,9 immunotype, of which 13% additionally expressed the L1,8,10 determinant. The LOS immunotypes of carrier strains were much more heterogeneous. The predominant immunotype was L1,8,10 (70%) and only 24% expressed L3,7,9 alone. Genotypically related case isolates from Norway (6) and Austria (18) expressed the L3,7,9 immunotype with similar frequency to the U.K. isolates. The combination of LOS immunotype and capsule expression appears to be related to the virulence of these meningococcal strains.


Conserved regulatory mechanisms of tyrosinase genes in mice and humans. In vertebrates, melanin production is restricted to pigment cells. This cell type-specific melanogenesis is considered to involve cell type-specific expression of the tyrosinase gene. Recently, there have been several reports that sequences in the 5' flanking region of the mouse tyrosinase gene are responsible for cell type-specific expression of the transgene in mice. As the first step in the study of the evolution of the regulatory mechanisms for tyrosinase gene function in vertebrates, we constructed a fused gene, hg-Tyrs-J, which includes a 1.0-kb 5' flanking sequence of the human tyrosinase gene fused with mouse tyrosinase cDNA. By introducing the fused gene into fertilized eggs of albino mice, we obtained two mice that exhibited pigmentation in the skin and eyes and established a transgenic line from one of them. Further analyses revealed that the transgene was expressed cell type-specifically in these transgenic mice. We conclude, therefore, that the 1.0 kb 5' upstream region of the human tyrosinase gene contains conserved cis-elements essential for cell type-specific expression of the tyrosinase genes in mice and humans. Results of our study may provide a clue to elucidate the evolutionary process of regulatory mechanisms of the tyrosinase gene.


Cleft lips and palates: a multidisciplinary approach. Families of infants born with congenital defects such as cleft lip, cleft palate, and combinations of these often grasp with accepting the condition. Nurses can help facilitate acceptance by parents and families and play a key role in the management and habilitation of these children.


A molecular semiotic view of biology. Interferon and 'homeokine' as symbols. A term "homeokine" was introduced as a generic name covering cytokines and protein hormones which serve the purpose of intercellular communication within the animal body for homeostasis and ontogenetic development. The homeokine system, in its complex way of functioning, seems to be analogous to another communication system, human language. Individual homeokine molecules are likened to words; they have meanings and are viewed as symbols, representing those conditions or events inside and outside the body which are relevant to homeostasis. Extending this view, any protein and other molecule can be considered to take on the character of sign, when integrated into a purposive system of higher hierarchy, because the molecule then represents a meaning relative to the system as a whole that is lacking in the isolated state. Living systems with their biological macromolecules as semantic units are constructed upon the principle of double articulation, just like human languages with words as the semantic units. The structure and function of a molecule (of protein and any other substance) are associated with each other, with various degrees of arbitrariness, as are the expression and the content of a sign in general. Namely the activities or the sign functions of biological molecules are determined by the organized system they belong to, and not vice versa.


Transurethral prostatectomy compared with incision of the prostate in the treatment of prostatism caused by small benign prostate glands. In a prospective, randomized study 60 patients with prostatism caused by small prostate glands (estimated weight < 20 g) had either transurethral prostatectomy (TURP, n = 31) or transurethral incision of the prostate (TUI, n = 29). Operating time and blood loss were significantly less in the group that underwent TUI. There were no differences between the groups in number of days with an indwelling catheter or days in hospital after operation. Eight patients in the TUI group required further operation, as did four in the TURP group, one of whom was discharged with a permanent indwelling catheter. In addition one patient developed a urethral stricture. Nine of the failures of treatment occurred within the first month. Fifty-one patients were followed up at 3 months and 47 were also seen at 12 months. Both operations significantly improved symptom scores and maximum flow rates compared with preoperatively, but the improvement in maximum flow rate was significantly better in the TURP group than in the TUI group. At 12 months TURP had also improved micturition time and voided volume, which TUI had not. Neither operation caused any significant change in sexual activity or erective potency postoperatively. Retrograde ejaculation was, however, seen in more than half of the patients in the TURP group, and only one in the TUI group. We recommend TUI for the treatment of prostatism caused by small prostate glands in patients who want to preserve normal ejaculation or are at poor surgical risk.


Transcervical prostatectomy in perspective. In a prospective study, 175 consecutive patients were included in transcervical prostatectomy. The results of the operation were evaluated by the mean weighted score and the postoperative morbidity. Although, urethral catheter was routinely removed on the second postoperative day, there was no evidence of suprapubic urinary fistula, clot retention or incontinence of urine noticed in any patient. Recatheterisation was required only in 2 patients for atony of the bladder muscles. Apart from wound infection in 3.4%, there was no other morbidity encountered with this technique. It is encouraging that about 77% of the patients went home before the fifth postoperative day and only one patient died on the second day. The merits of this technique have been highlighted in perspective with other studies reported in the literature.


Developing a poster for disseminating research findings. One of the goals of research should be the dissemination of research findings. Presentation of research findings to an audience of interest can be accomplished through formal or informal presentations at professional conferences or on a departmental level, publication in a variety of professional journals, and presentation as a poster session. Poster presentations attract an audience of similar interest, permit interaction and clarification of the research process and findings by the researcher, allow creative expression in presentation, and help to build collegial relationships with others who hold similar interests. Posters are also a way for educators to present the research findings of others that answer specific clinical questions or provide a means of staff development. A well-designed poster gives the viewer a concise overview of the research problem/purpose, research methodology, sample, results, and implications. Since the less formal poster presentation is viewed as less intimidating to some researchers and conference participants, posters may provide a more relaxed atmosphere in which the exchange of information takes place.


[Changes in antiproteolytic activity in the blood and cervical mucus after superovulation stimulation]. The function of proteins, peptides, proteases and inhibitors of proteases in modulations of regulation mechanisms of gonadotrophins during the development of ovarian folicles has not been fully explained up to now. We can see difference reactions of ewes to superovulation stimulations in oestrous and anoestrous periods as shown by the variation of the antiproteolytic activity of blood plasma and cervical mucus. Trypsin is used as a model for serine protease, and trypsin inhibitory activity (TIA) was measured from the reduced rate of trypsin hydrolysis of the chromogenic substrate N-alpha-tosyl-L-arginine-4-nitroanilide (TAPA, Bartík et al., 1974). Full hydrolytic activity was determined as a change in absorbency at 405 nm = 1.0 after ten-minute incubation at 25 degrees C and pH = 8.1, and inhibition was expressed in percentage of full activity. Statistical analyses were performed by Student's t-test. Twenty-three ewes in anoestrus and twenty-eight ewes in oestrus were included in this experiment. They were of the Slovak Merino breed, two to three years old, with the mean live weight of thirty to forty kg. The ewes were treated with Ageline vaginal sponges (20 mg chlorsuperlutin/sponge) to provide for synchronization of ovarian activity, in the interval of eleven to twelve days. After removal of sponges, the ewes were stimulated for superovulation as shown in Tab. I. Heparinized blood plasma samples were stored at -25 degrees C. Low molecular TIA activities were determined in HClO4--treated blood plasma. Samples of cervical mucus were taken on cotton-wool tampons which were evaluated in 0.2 M Tris-HCl buffer, pH = 8.1. Figs. 1-6 shows TIA changes in blood plasma and cervical mucus on particular days. Fig. 7 shows the mean values of controls (I-initial) after synchronization (A) and after stimulation (S). TIA changes were different in anoestrous and oestrous periods. Differences in TIA changes in blood plasma and cervical mucus in oestrous period suggested certain local regulation mechanisms of synthesis and/or secretion of this activity in the cervix. Some values of TIA were maximum at the time of expected heat or ovulation and may be some of the factor(s) which influenced fertility of females after superovulation stimulations.


Cerebellar connections of the ventral lateral geniculate nucleus in the rat. The connections between the deep cerebellar nuclei and the ventral lateral geniculate nucleus (LGv) were investigated in rats using orthograde and retrograde transport of horseradish peroxidase. Following injections into the deep cerebellar nuclei there was orthograde transport to the contralateral medial LGv and adjacent zona incerta. Injections restricted to LGv consistently labelled a small cluster of cells in the contralateral posterior interposed nucleus. Injections into regions surrounding LGv produced distinctively different patterns of orthograde and retrograde labelling.


Cytokinetic studies on the aortic endothelium and limb bud vascularization in avian embryos. Cytokinetic studies on the aortic endothelium using the BrdU/anti-BrdU-method were carried out on 2.5- to 6-day chick and quail embryos. The mitotic activity of the aortic endothelium is related temporally to the age of the avian embryo and spatially to the embryonic region where the aorta originates. The mitotic activity of the aortic endothelium decreases with increasing age of the embryos. In the limb buds, however, the mitotic rate of the aortic endothelial cells increases independently of the age of the embryo. This increase in the mitotic activity of the aortic endothelium at the appropriate levels coincides with the vascularization of the outgrowing limb buds. We concluded therefore that the aortic endothelium probably supplies endothelial cells for the formation of limb vessels at this stage. Thus our results suggest that angiogenesis (sprouting of capillaries from pre-existing vessels) takes place during limb vascularization in avian embryos. On the other hand, immunohistochemical studies with QH-1 or MB-1 antibody show, beside a capillary network in the central core of the wing bud, individual immunolabelled cells of mesenchymal character within the primarily avascular subectodermal region from the onset of vascularization onwards. We suggest that these cells have partly to be regarded as endothelial precursor cells, which have differentiated in situ from the local limb mesenchyme, and which will contribute to the developing vascular plexus. This means that not only angiogenesis, but also vasculogenesis (in situ from mesenchymal precursors differentiated endothelial cells) appears to be involved in limb vessel formation.


The effect of saffron on intracellular DNA, RNA and protein synthesis in malignant and non-malignant human cells. Extract of saffron (Crocus sativis) has previously been shown to inhibit colony formation and cellular DNA and RNA synthesis by HeLa cells in vitro. In order to compare the sensitivity of malignant and non-malignant cells to saffron, we examined the effect of the extract on macromolecular synthesis in three human cell lines: A549 cells (derived from a lung tumor), WI-38 cells (normal lung fibroblasts) and VA-13 cells (WI-38 cells transformed in vitro by SV40 tumor virus). We found that the malignant cells were more sensitive than the normal cells to the inhibitory effects of saffron on both DNA and RNA synthesis. There was no effect on protein synthesis in any of the cells.


Scientific comment. Basic science of Dupuytren's disease. At a cellular level, the major phenomenon in Dupuytren's contracture is an increase in proliferating fibroblasts, rather than an alteration in the type of fibroblasts. The collagen and glycosaminoglycan changes observed in the tissues of Dupuytren's contracture are likely to be secondary to changes in fibroblast density. Oxygen free radicals released from narrowed microvessels and from the fibroblasts themselves may play a role in stimulating fibroblast proliferation. Messina's experiments with continuous distraction and the success of limited fasciotomy indicate a role for physical forces in the pathogenesis of Dupuytren's contracture. The mechanism whereby physical events stimulate biochemical changes are undetermined, but preliminary investigations suggest that electrical fields and ion channels may be important. The future of Dupuytren's contracture rests with the delineation of these chemical and physical processes and their application and rigorous testing in the clinical setting.


Open palm technique for Dupuytren's disease. A five-year follow-up. One hundred and seven patients presenting 140 localizations of Dupuytren's disease were operated upon by a single surgeon before 1985, according to a modified Mac Cash procedure combining a Bruner approach and open transverse digital and/or palmar incisions. All surgery was performed under axillary block on an out-patient basis. A striking feature is the absence of complications such as haematoma, skin necrosis or infection. In the early post-operative follow-up (mean 105 days), the average angular improvement was 79.5% in all patients. Among the 107 patients, 54 (for a total of 67 localizations) were specifically reviewed with an average follow-up of 5.6 years. Improvement averaged 74% in 83.5% of digits. In the remaining 16.5%, the lack of extension averaged 31 degrees. Some factors had a negative effect on final results: early age of onset, major involvement of the PIP joint, localization at fifth ray level. The recurrence rate (41%, 23% of which severe) is identical to other reports on limited fasciectomy. A good indication is therefore a patient older than 50, and the method provided low postoperative morbidity and pain, associated with acceptable results.


A natural history of Dupuytren's contracture treated by surgical fasciectomy: the influence of diathesis (76 hands reviewed at more than 10 years). Fifty-six patients (51 males and 5 females) were reviewed more than 10 years after surgical fasciectomy for Dupuytren's disease (average 12 years 7 months). 76 hands were operated upon (20 bilateral cases) for a total of 137 fingers (out of 152 affected digits). The average pre-operative score based on Tubiana's classification was of 4.28. To-date, at maximum follow-up time, it is of 2.45. 34 recurrences occurred before re-operation (i.e. 45%), whereas at maximum follow-up, 74% of hands are still defective, although only 22% cause functional impairment. The method of improvement ratio classifies patients into two categories: satisfactory results and failures. This original method of qualification emphasizes three negative factors: recurrences, initial severity of disease, and failure to achieve full finger extension post-operatively. Results as a whole are in accordance with those mentioned in literature. Improved results depend on early surgery for severe cases, which must therefore be diagnosed at an early stage. Surgery often sets back the disease, without curing it. Further efforts in fundamental research on the etiology of this disease will be needed.


A mid-term review of the results of dermofasciectomy for Dupuytren's disease. Clinical involvement of the palmar and digital skin by Dupuytren's disease occurs frequently. A review of 40 rays in 32 patients who had consecutively undergone dermofasciectomy is presented. The average follow-up period was 38 months, with a minimum of 24 months. Four out of the 40 rays developed recurrent nodule formation limited to the proximal or distal graft insets but there was no recurrent cord formation, suggesting better disease control than by fasciectomy alone. Full thickness graft to resurface the defect has been surprisingly complication-free.


Dermofasciectomy: a long term review.

Dermofasciectomy: a long term review. Thirty-two dermofasciectomies in 24 cases were reviewed with an average of thirteen years for follow-up. These were compared with the same patients followed-up ten years earlier. A marked increase in extension of the disease from 24% to 79% was noted. The recurrence rate has remained constant at 47% and only two recurrences were found under the skin graft. This procedure must, therefore, remain the operation of choice in the management of aggressive Dupuytren's disease.


Compartment syndrome of the scapula. Definition on clinical, neurophysiological and magnetic resonance data. Compartment syndromes of the scapula and pelvic girdle have received scant attention in the literature. In 1938, Comolli first described a clinical sign which he considered specific to fracture of the scapula. We report data on two patients, one presenting with prolonged pressure on the posterior surface of the scapula and the other with symptoms associated with scapular fracture. In one of these patients we were able to measure pressures around the scapula, perform neurophysiological assessment of nerve function and produce magnetic resonance images of the area. In the other case, surgical exploration revealed an established ischaemic contracture of the infraspinatus muscle within its compartment. These findings suggest that the muscles around the scapula are vulnerable to the development of compartment syndrome.


Ultrasonography of the normal and abnormal antenatal development of the upper limb. The degree of severity of congenital deformities of the hand, as well as any coexisting congenital anomaly should be evaluated especially if there is a possibility of therapeutic termination of pregnancy. This may be relatively easy in the case of some major malformations which affect the neurological or intellectual capacity of the child. It is however more difficult in the presence of predominantly motor anomalies, and with distally based anomalies of the upper limb which may be amenable to later reconstructive surgery.


Forty-eight glomus tumours of the hand. Retrospective study and four-year follow-up. We report a series of 48 glomus tumours of the hand, which have been reviewed with an average follow-up of 4.5 years (2-14). The tumour was nearly always sited in the distal digit, para-ungual (22 cases), more rarely subungual or under the pulp, with an even distribution among the fingers. Diagnosis was made preoperatively in 37 of 47 tumours of the digits, based on clinical features: consistent eliciting of pain by touch, less often by chill and occasionally accompanied by vasomotor phenomena. The pain and therefore the tumour could be accurately located with the tip of a pencil (Love's sign); these symptoms are abolished by inflation of a tourniquet proximally (Hildreth's sign). In 40% of cases a small defect in the distal phalanx is visible on plain radiographs, as well as an enlargement of the subungual tissues when the tumour is dorsal. The surgical approach was usually para ungual, in a sub-periosteal plane. Tumours were small (3.3 mm) and rarely multiple (3 cases). In 46 cases the pain was relieved quickly and definitively. Two true recurrences occurred after 5 years, without a satisfactory explanation. Except for those cases with transungual approach, there was no aesthetic compromise. We maintain that careful clinical assessment provides the diagnosis in most cases. Plain radiographs, lateral and comparative, are useful. MRI scan may occasionally prove of diagnostic value. The lateral ungual approach permits complete excision and healing. Recurrence is rare.


Idiopathic avascular necrosis of the scaphoid. A case report. A single case of non-traumatic, idiopathic avascular necrosis of the carpal scaphoid is presented. It permits an informative correlation of clinical evolution. X-ray appearance, MR-imaging, macroscopic aspect and histological findings.


Combined second toe and partial nail transfer from the big toe by means of an exteriorized pedicle. The hand of a three-year old girl was reconstructed by means of a bilateral second toe transplantation following traumatic amputation of all five digits. In a second operation, aimed at reconstructing the index finger, the nail apparatus of the big toe was transferred to the thumb as an "island flap" based on a pedicle from the second toe. The thumb was subsequently lengthened enabling a three-fingered hand to be obtained with only minimal functional impairment of the feet. The authors suggest an original technique allowing transfer of the second toe and of the big toe nail in only one operative stage. The technique of leaving the pedicle exteriorized during transplantation (autonomization) facilitates positioning of the nail onto the recipient site and eliminates subsequent problems of commissural retraction.


A palmar plaque and flexor tenosynovitis in a patient with sarcoidosis. Involvement of the soft tissues of the hand by sarcoidosis is rare. We report a patient with sarcoidosis who presented with a large subcutaneous plaque of the palm and flexor tenosynovitis of the middle finger.


The ubiquitous transactivator Zfp-38 is upregulated during spermatogenesis with differential transcription. We describe the complete nucleotide sequence of a full length cDNA clone encoding a new mouse zinc finger protein gene, Zfp-38 and localize it on chromosome 5 by the interspecific backcross analysis. The N-terminal domain of the Zfp-38 protein (64 kDa) contains 358 amino acids and the C-terminal domain of 197 residues encodes 7 zinc fingers. We also present evidence that Zfp-38 is a strong transcriptional activator. The transactivation domain was localized in the non finger region and a fusion protein containing 112 amino acid residues from this region of the Zfp-38 and the DNA binding domain of the yeast Gal 4 protein, very efficiently transactivated the expression of a reporter CAT plasmid, harboring the Gal4 target site. By in situ hybridization and northern blotting technique, the Zfp-38 transcript can be detected at a highly elevated level during spermatogenesis. Its expression accompanies the progression from pachytene spermatocytes to round spermatids. The undifferentiated spermatogonia or the haploid elongated spermatid and the spermatozoa do not show any detectable level of the transcript. Interestingly, other tissues express low levels of a slightly shorter transcript with a different 5' end as determined by RNase protection. The presence of both a transcriptional activating domain and 7 DNA binding zinc fingers, coupled with the cell type(s) specific expression pattern, suggests that Zfp-38 has the potential to regulate transcription during spermatogenesis.


The cloning and characterization of a localized maternal transcript in Xenopus laevis whose zygotic counterpart is detected in the CNS. We have cloned a cDNA (xlan4) from a Xenopus laevis oocyte cDNA library whose cognate mRNA is localized in the animal pole region of full grown oocytes. The cDNA can be translated in vitro to produce a predicted size protein of 35 kDa and, is also expressed in E. coli as a fusion protein. The conceptual protein encoded by the xlan4 cDNA is 17.5% proline rich and possesses several PEST sequences found in proteins with short half-lives. The xlan4 mRNA is 2.6 kb and during early development its titer decreases until the neurula stage after which it begins to reaccumulate. Northern blots on dissected embryos and in situ hybridization revealed that the zygotic expression is limited to the dorsal axial structures consisting primarily of the CNS. UV irradiation of the vegetal pole region immediately following fertilization that produces ventralized embryos results in a loss of zygotic xlan4 expression. In the adult, xlan4 mRNA is limited primarily to the brain. The presence of this mRNA in animal pole region which contributes to the future neural cell lineages suggests that this gene product may function either in the specification of neural cell types or in a neural specific function.


Effects of calcium channel agonism by Bay-K-8644 on ventricular fibrillation threshold of isolated heart. The hypothesis tested was that enhanced entry of calcium into cardiac cells would increase the susceptibility to ventricular fibrillation as measured by the ventricular fibrillation threshold (VFT) of the isolated perfused rat heart. Bay-K-8644 was used as a calcium-channel agonist. There was a biphasic effect with a maximal increase in left ventricular systolic pressure and oxygen uptake at a concentration of 10(-7) M. The same concentration caused a major reduction in the VFT. The bell-shaped pattern of fall of the VFT was inversely related to the effect on LV developed pressure. The changes in VFT could be dissociated from those on myocardial metabolites. Although Bay-K-8644 increased the heart rate, reduction of the VFT could also be obtained in paced hearts. The addition of ryanodine, an agent known to interrupt intracellular recycling of calcium through the sarcoplasmic reticulum, was able to abolish approximately half the effect of Bay-K-8644 on the VFT. Therefore, increased entry of calcium via the calcium channel is able to reduce VFT, acting in part through enhanced recycling of calcium through the sarcoplasmic reticulum.


Collateral formation after repeated transient dearterialization of the rat liver. Hepatic artery ligation is used for the palliation of patients with malignant liver tumours. Collaterals are developed rapidly and could to some extent explain why the growth is affected for only a short period. With intermittent dearterialization, collaterals seem to be avoided and possibly a more extended effect should be expected. The most efficient period of dearterialization to avoid collaterals was studied in this experiment. Five groups of rats were treated with daily repeated transient dearterializations for 0 (n = 3), 60 (n = 6), 120 (n = 6), 180 (n = 6) and 240 minutes (n = 6) respectively for 5 days and compared to another group (n = 3) that was permanently dearterialized. After treatment, celiac angiograms were obtained. All hepatic arteries were reliably occluded and patent after 5 days of daily blockades in all but two rats. There were no collaterals demonstrable on the angiograms in the first four groups after 5 days of intermittent obstruction of the arterial blood flow to the liver. After 240 minutes of dearterialization as well as after collaterals developed and were clearly demonstrated on the angiograms after six days. Liver enzymes were normal even after 4 hours of dearterialization. Repeated occlusions of the hepatic artery was reliably achieved with the implantable minioccluder. Repeated, transient dearterializations for 1, 2 or 3 hours could be performed without development of collaterals and without damage to the liver.


Pulmonary injury in acute experimental pancreatitis correlates with elevated levels of free fatty acids in rats. Since some authors have stated a certain role for so-called "free fatty acids" (FFA) in the pathogenesis of AP and the subsequent systemic complications we tried to find possible correlations between FFA, pancreatitis and lung injury using a rat model. AP was induced by intraductal infusion of two different concentrations of glycodeoxycholic acid (GDOC 17 mmol and 34 mmol). An equal number of animals had only cannulation of the pancreatic duct without infusion and served as controls (GDOC-control). In another experimental model iv.-infusion of oleic acid (OA) was used to create severe lung injury comparable to human ARDS. In this model control animals received iv.-infusion of saline solution only (SAL). At 2, 6, 12, 24 and 48 hours the animals were sacrificed and blood was collected for determination of FFA, amylase and pO2. The pancreas and lungs were removed for histologic examination and the lungs were weighed. GDOC-34 animals developed severe pancreatitis with hemorrhage and necrosis. Histology of the lungs showed edema, inflammatory infiltrates, hemorrhage and thickening of the alveolar membrane in GDOC-34 rats as well as in OA-animals. In contrast, there was only pancreatic edema until 24 hours in the GDOC 17 group and less severe histological changes in the lungs. Amylase, FFA, pO2 and lung weight were directly influenced by the different kinds of treatment. Furthermore, FFA correlated positively with the levels of amylase and lung weight and negatively with pO2. Infusion of OA alone also caused an increase in levels of amylase with pancreatic edema and focal necroses in some animals. These results show that it was possible to create different degrees of severity of AP which was in concordance with different degrees of morphologic changes and dysfunction in the lungs. FFA values correlated significantly with the clinical course as well as with increasing amylase, lung weight and decreasing pO2.


[Bleomycin emulsion for intralesional instillation in advanced oropharyngeal carcinomas. A case report on local bleomycin application]. Local therapy of head and neck cancer using a special bleomycin emulsion as an intratumoral applicarted cytotoxic agent is not established in regular antitumor therapy. It's use only is supposed to be regarded as an "ultima ratio" possibility when other usual methods like surgery, radiotherapy or systemic cytostatic therapy have failed. The procedure of intralesional bleomycin emulsion application will be described and the advantages and disadvantages will be discussed by means of a long-term case report.


Contrasting effects of interferon gamma and interleukin 4 on responses of human vascular endothelial cells to tumour necrosis factor alpha. Tumour necrosis factor alpha (TNF-alpha) and interleukin 4 (IL-4) selectively synergise in inducing expression of the mononuclear cell adhesion receptor VCAM-1 (vascular cell adhesion molecule-1) on human umbilical vein endothelial cells (HUVEC), which results in increased adhesiveness of HUVEC for T lymphocytes. This process may be crucial for adherence of circulating lymphocytes prior to their passage from the blood into inflammatory tissues. IL-4 also amplifies production of interleukin 6 (IL-6) and monocyte chemotactic protein-(MCP-1) from TNF-alpha-activated HUVEC. In the present study we demonstrate that IL-4 enhances production of granulocyte-macrophage colony-stimulating factor (GM-CSF) from TNF-alpha-stimulated HUVEC. Moreover, using cultured adult saphenous vein and umbilical artery endothelial cells, we show identical effects of IL-4 on TNF-alpha-induced responses to those observed with endothelial cells of foetal origin. Additionally, we report here that TNF-alpha and interferon gamma (IFN-gamma) synergise in the induction of both the lymphocyte adhesion receptor VCAM-1, and the TNF-alpha-inducible neutrophil adhesion receptor intercellular adhesion molecule-1, on all three endothelial cell types studied. In contrast, we found that GM-CSF secretion by endothelial cells treated with IFN-gamma plus TNF-alpha was markedly decreased when compared to the response induced by TNF-alpha alone. These results suggest that the combined actions of several cytokines, acting sequentially or in concert, may exert differential effects on activation and accumulation of circulating lymphocytes at sites of inflammation.


Derivation of a monoclonal anti-murine IL-4 antibody specific for an epitope expressed on denatured molecules. Monoclonal antibodies to interleukin 4 (IL-4) were generated by immunization with recombinant IL-4 and screening by binding to IL-4 adsorbed to plastic surfaces. Three antibodies were obtained that scored well in this assay and one, 13E1, was studied in detail. It was very effective in detecting IL-4 by Western blotting whereas a neutralizing anti-IL-4 antibody, 11B11, was 50-100-fold less sensitive as a detecting agent. Sequential immunoprecipitation and biosynthetic labelling studies indicated that the 11B11 and 13E1 epitopes are largely expressed on different forms of IL-4. 13E1 was able to detect cytosolic IL-4 both by immunohistochemical and flow cytometric analysis of fixed cells. This was routinely successful in an insect cell line (Sf9) expressing large amounts of IL-4 as a result of infection with a recombinant 'IL-4 baculovirus'. Although stimulated D10 cells could also be shown to express IL-4 in their cytosol, positive results were not obtained in all such studies and we have failed to detect IL-4 production by normal T cells using this method. This antibody may have substantial value in detecting IL-4 by Western blots and as a tool to analyze the biosynthesis of IL-4. With suitable improvement in sensitivity, it also may prove valuable in the detection of IL-4 in the cytosol of individual cells.


Serum granulocyte colony-stimulating factor levels in umbilical cord blood of normal full-term neonates. We measured granulocyte colony-stimulating factor (G-CSF) levels in cord blood of 59 normal full-term neonates immediately after birth and the subsequent changes in G-CSF levels of 16 cases by our modified enzyme-linked immunosorbent assay (ELISA) for G-CSF. Ten out of 59 cases examined (17%) showed G-CSF levels in cord blood after delivery between 20 and 57 pg/ml, although in the remaining cases, the G-CSF levels were below 20 pg/ml, which was our minimal detection level. A direct relationship between G-CSF levels and white blood cell count, absolute granulocyte numbers in cord blood, gestation age or weight was not observed. Although G-CSF levels in cord blood after delivery remained below 20 pg/ml in 5 cases out of 16 tested, those in the remaining 11 cases (69%) subsequently became elevated after delivery. The peak G-CSF level in cord blood after delivery ranged from 26-364 pg/ml, and the time of it was between 4.5 and 18 h. As G-CSF level per wet weight of placenta was high (124 x 29 pg/ml), these subsequent elevations of G-CSF in cord blood after delivery may result from a gradual influx from the placenta.


[Monoaminoxidase activity and serotonin metabolism in the brain of rats during latent inhibition]. The monoamine oxidase (MAO) activities and the concentrations of 5-HT and 5-hydroxyindoleacetic acid were investigated in four brain regions in rats during the acquisition of latent inhibition in one-trial passive avoidance task. 5-HT metabolism was not altered in the hippocampus. Changes of 5-HT metabolism were found in the frontal cortex during testing of latent inhibition and were accompanied by lowering of MAO activity. No change of 5-HT metabolism was observed in this structure at the stage of pre-exposition to conditioned stimulus. 5-HT metabolism was activated at the stage of pre-exposition to conditioned stimulus in the amygdala and striatum and was maintained on high level, in these structures during testing of latent inhibition. The data presented here indicate that serotoninergic system in various brains regions is specifically involved in the formation of different stages of latent inhibition.


[Emoxipin as an inhibitor of angiogenesis]. The effect of emoxypin on angiogenesis in rabbit cornea in aseptic inflammation induced by intracorneal implantation of a piece of quartz and on the development of the vessels of the chick embryo yolk sac was studied. 1% emoxypin pipetted thrice a day for 10-14 days inhibited corneal neovascularization and reduced the formation of new blood vessels. We observed an inhibitory effect on the development of vascular bed of the embryo yolk sac on incubation hour 64-72. The drug affected neither general growth of the embryos no the number of somites.


[The immunodiagnosis of thrombocyte membrane glycoprotein deficiencies: Glanzmann's thrombasthenia, Bernard-Soulier syndrome and GMP-140 protein deficiency]. Immunological methods were developed for the diagnosis of platelet membrane glycoprotein (GP) deficiencies. The number of membrane GP on platelet surface was determined as the binding of 125I-labeled monoclonal antibodies (mAB) directed against individual platelet GP. Total amount of GP in platelet lysate was assessed by immunoblotting with specific polyclonal antibodies. Methods were applied for patients with different thrombocytopathies. Binding of mAB VM16a, directed against GP IIb-IIIa was strongly decreased in patients with Glanzmann's thrombasthenia (0.5-14.5% of normal) and binding of anti-GP Ib mAB VM16d--in patient with Bernard-Soulier syndrome (0.5% of control) indicating the deficiencies of corresponding GP. In patient with gray platelet syndrome binding of both antibodies was not decreased but even increased. It was shown by immunoblotting that platelets from the patient with gray platelet syndrome contained normal amount of GP IIa, but strongly decreased amount of GMP-140 (14.5% of control)--membrane GP of platelet--granules.


The zonal expression of alpha-fetoprotein transgenes in the livers of adult mice. The developmental regulation of the alpha-fetoprotein (AFP) gene in liver results in high-level expression in the fetus, followed by dramatic transcriptional repression after birth. We have examined the mouse AFP gene for transcriptional control sequences that may be involved in its postnatal repression in liver. We showed previously that removal of a DNA region between positions -250 base pairs (bp) and -838 bp of the AFP gene resulted in the persistence of expression of an AFP minigene in the postpartum liver of transgenic mice (Vacher and Tilghman, Science 250:1732-1735, 1990). This study examines the distribution of these transgene transcripts in liver using in situ hybridization. We show that there is a zonal distribution of minigene transcripts in the adult livers of these animals. Hepatocytes surrounding the central veins express high levels of minigene transcripts, while hepatocytes in the intermediate and portal areas contain few, if any, transcripts. Quantitative RNAse protection analysis shows a decrease in transgene RNA levels after birth, consistent with repression in all but a small subset of hepatocytes. These results indicate that repression in the pericentral hepatocytes is dependent upon the presence of a cis-acting, negative-regulatory domain, which is located between the enhancers and the proximal promoter of the AFP gene. In contrast, this domain is not essential for complete repression of AFP transgenes in the intermediate zone and periportal hepatocytes.


[Prognostic factors, late relapse and outcome of patients with aggressive malignant lymphoma: long term results of LNH-80 protocol]. The 100 patients with aggressive malignant non Hodgkin lymphoma treated with the LNH-80 regimen were evaluated for long-term survival and quality of life. The LNH-80 regimen consisted of three intensive courses of adriamycin, cyclophosphamide, vindesine and bleomycin, followed by consolidation and final intensification, as previously described. Of the eighty-four patients who achieved CR after induction, fifty-two patients are alive in continuous complete remission with a median follow-up of 9.2 years. Twenty-nine CR patients (35%) relapsed. Sixty-six percent of the relapses occurred during the first 18 months following the end of treatment but late relapses were observed up to 7 years off-therapy. Factors adversely correlated with response in multivariate analysis were poor performance status, bone marrow involvement and the presence of more than one extranodal site. Factors adversely correlated with survival were age, high grade subtypes, and bone marrow involvement. The fifty-two long-term responders were evaluated for quality of life parameters such as working ability, sexual function, fertility and absence of long-term sequelae. Of the 41 patients who worked before disease, 66% had resumed their normal job and 24% had retired. Sexual activity was considered to be satisfactory by 66% of the patients. Eleven of the 15 patients (73%) who wanted children had between one and three children. Seven patients (14%) considered having mild long-term sequelae but all long-term survivors reported having an acceptable quality of life. Five of the patients who reached CR (6%) had a second neoplasia. The LNH-80 regimen allowed 52% of the patients to benefit from long-term disease-free survival with minor long-term toxicity.


Poly(L-lysine)-conjugated oligonucleotides promote sequence-specific inhibition of acute HIV-1 infection. Previously, we have reported that conjugation of antisense oligonucleotides to poly(L-lysine) (PLL) lowers their inhibitory concentration in several biological models. We have now tested these conjugates for inhibition of human immunodeficiency virus type 1 (HIV-1) replication. PLL-conjugated oligonucleotides complementary to the translation initiation site of Tat protein protect cells from the cytopathic effect of HIV-1 in acute infection assays. The EC50 of conjugates is approximately 0.15 microM, which represents a strong reduction in concentration as compared to nonconjugated oligonucleotides (EC50 = 20 microM). In contrast with most reports in the literature, we have observed sequence specific antiviral effects with PLL conjugates. This was particularly noteworthy in antiviral experiments performed with HIV-1 isolates presenting heterogeneity in the 5' end of the tat mRNA sequence. Two mismatches at the target site were sufficient to reduce very significantly the antiviral activity of the conjugates but did not modify the effect of nonconjugated oligonucleotides. Unlike free oligonucleotides, PLL-conjugated ones do not interfere with virus penetration and/or reverse transcription as demonstrated by polymerase chain reaction (PCR) analysis of viral DNA.


Potentiation of radiation lethality in mouse melanoma cells by mild hyperthermia and chloroquine. To test the hypothesis that radiosensitization by combined mild hyperthermia and chloroquine may be increased by the presence of melanin in treated cells, Cloudman melanotic mouse melanoma S91/6 cells, and the amelanotic S91/amel cells were incubated during a 3 h post-irradiation period with 0.03 mM chloroquine at 41 degrees C. A considerable increase in radiation lethality was observed (radiation potentiation factor > 1.6) in both cases. Addition of 0.1 mM isobutyl-methyl xanthine (IBMX), a promoter of melanin synthesis, to the growth medium of S91/6 cells 10 days before irradiation, did not further increase the lethality of radiation followed by combined heat and chloroquine treatment. Under these conditions, toxicity to unirradiated cells was slight. On the other hand, 10 microM chloroquine showed similar toxicity to unirradiated B-16 mouse melanoma cells, but did not increase radiation lethality. Factors other than melanin content therefore play a role in the potentiation of radiation lethality by mild hyperthermia and chloroquine.


[Effect of immunosuppressants FK506 and rapamycin on the function of progesterone receptor: protein "p59-HBI", intersection between immunology and endocrinology?]. In the absence of hormonal ligand, inactive, heterooligomeric, 8-10S steroid receptor complexes include a p59 protein (apparent M(r) approximately 59 kDa) bound to th heat shock protein hsp90 (apparent M(r) approximately 90 kDa), which itself binds to the ligand binding domain LBD of the receptor molecule, p59 is thus an hsp binding immunophilin HBI, which, through its interaction with a chaperone, may intervene in several cellular functions. We report that, in cell-free experiments at 0 degrees C, FK506 and rapamycin do not release p59 nor hsp90 from the 9.5S rabbit uterus progesterone receptor, suggesting that the binding of p59 to hsp90 does not interfere with the rotamase site of HBI. There is no "transformation/activation" of the receptor, but an up to 2 fold increase in progesterone agonist and antagonist binding to the receptor is observed. It is suggested that a functional interaction between HBI and receptor activity may be mediated by hsp90.


The application of immunocytochemistry in diagnosis of meningeal carcinomatosis in a patient with unknown primary site. A 55-year-old female with meningeal signs was suspected to have carcinomatosis meningitis based on cytospin cytology study of cerebrospinal fluid (CSF) with Wright stain. There was no primary site of any malignancy which could be identified as the source of metastasis. Immunochemical staining for cytokeratin and carcinoembryonic antigen on suspicious large immature cells in the CSF gave positive results and confirmed the malignant nature of her disease. Intrathecal chemotherapy with methotrexate and whole brain irradiation then eradicated the symptoms rapidly. Immunocytochemistry was considered to be a very powerful diagnostic tool in management of this patient.


Systemic and regional oxygen uptake and lactate flux in endotoxic dogs resuscitated with dextran and dopexamine or dextran alone. We examined how volume resuscitation with and without dopexamine, a potent beta 2-adrenergic and dopaminergic agonist, would affect O2 uptake (VO2), O2 delivery (DO2), and lactate metabolism in whole body, skeletal muscle, and gut of 18 anesthetized dogs after they were infused for 1 hr with 2 mg/kg endotoxin. Six dogs were then infused with dextran for 2 hr at 0.5 ml/kg/min (DEX); another six were given the same volume with 12 micrograms/kg/min dopexamine (DPX); and a third group received no treatment (NT). After endotoxin, all animals were hypotensive; cardiac output decreased 30%; and DO2 to whole body, skeletal muscle, and gut were at or below critical for VO2. DO2 was raised above critical in whole body and gut of DEX and DPX but not in muscle of DPX, which produced lactate. Whole body DO2 approached preendotoxin level by 2 hr in DEX and DPX but stayed near critical in NT. At that time, gut VO2 was higher in DPX than in DEX or NT, and lactate efflux became less than in DEX. Vascular reactivity was present in the gut at the end of the experiment in six of six DPX studies, in four of six DEX studies, and in none of the NT studies. These results suggest that resuscitation with dopexamine + dextran after induction of endotoxic shock may be more beneficial than dextran alone, particularly for the gut mucosa.


Inhibition of histamine release and pressure increase induced by endothelin (ET-1) in isolated rat kidneys treated with propionyl carnitine. Endothelin-1 (ET-1) is an amino peptide produced by endothelial cells with a potent vasoconstrictor activity; this effect is regulated by a release of other endogenous vasal factors. Recent studies have demonstrated that endothelin is capable of releasing from different tissues, and particularly from perfused organs such as spleen and kidney, many vasal factors and prostanoids. The present study investigates whether perfusion with a solution of endothelin-1 in rat isolated kidney can induce the release of histamine, another vasal factor (until now not investigated in relation with endothelin-1) and whether treatment with propionyl carnitine, a compound with vasoprotecting activity, inhibits this release. This research demonstrates that histamine released by rat kidney perfused with endothelin is lowered if previously treated with propionyl carnitine. This effect of propionyl carnitine can be considered to be another important factor in a complex mechanism involved in its vasoprotective and cardiovascular action.


Chemotherapeutic approaches to human immunodeficiency virus infections. Since the discovery of human immunodeficiency virus (HIV) as the causative agent of acquired immune deficiency syndrome (AIDS), various attempts have been made to control this fatal disorder. In the replicative cycle of HIV, several steps have been identified as attractive targets for antiviral chemotherapy. Sulfated polysaccharides can block the virion binding to the CD4 receptor. 2',3'-Dideoxynucleosides including 3'-azido-3'-deoxythymidine (AZT) act as potent inhibitors of reverse transcriptase after intracellular phosphorylation. Only AZT and 2',3'-dideoxyinosine are available so far for the treatment of AIDS and AIDS-related complex. Non-nucleoside reverse transcriptase inhibitors of HIV-1 and viral protease inhibitors are the new classes of compounds that are now extensively studied. These compounds may add a new dimension to the prospects of anti-AIDS chemotherapy.


False positive reaction of heat-inactivated sera in enzyme-linked immunosorbent assay for antibody to hepatitis C virus. Specimens were heated at 56 degrees C or 62 degrees C to analyze the mechanisms of false positive reactions in the Ortho enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to hepatitis C virus (HCV). The highest correlation was obtained between the difference in HCV antibody titer of 56 degrees C-heated and native sera and IgG concentration. Monoclonal IgG gammopathy sera developed marked increases in the titer by preheating. However, slight increases after preheating were observed in monoclonal IgA gammopathy sera. Similar increases in the titer by preheating were also demonstrated in immunoglobulin products containing IgG. Direct bindings of denatured IgG with C100-3 and 5-1-1 antigens were shown in recombinant immunoblot assay (RIBA). These results could show the participation of denatured IgG in the false positive reaction in Ortho-HCV-ELISA. Caution is necessary in evaluating the anti-HCV antibody titers of heat-inactivated sera.


Management of malignant germ cell tumours of ovary. Ten patients of the advanced malignant germ cell tumours of the ovary were treated by cisplatin based combination chemotherapy after initial conservation surgery. Eight patients completed course containing cisplatinum, vinblastine and bleomycin. Five patients (62.5%) achieved CR while 2 (25%) attained PR. One patient died due to tumour lysis and respiratory infection. Rest two patients did not turn up in follow up. Long term follow up indicates above regimen to be highly effective. However poor performance status, advanced stage of disease and post operative gross residual disease were poor prognostic factors in our patients.


Alpha-fetoprotein production by rhabdomyosarcoma of the urinary bladder. Alpha-fetoprotein has emerged as a useful diagnostic tool for hepatic tumour and tumours of germ cell origin. However, isolated case reports of association of this tumour marker with tumours of the lung and non-germ cell tumours of the ovary are reported. We present a case of a Rhabdomyosarcoma, a generally non-secretory tumour that showed raised levels of AFP in serum and reacted positively for the same in a PAP technique for AFP. Cross striations were visible in many cells on II & E stained sections.


Fine needle aspiration cytology in the diagnosis of prostatic enlargements. One hundred patients with enlarged prostate were subjected to fine needle aspiration cytology and histopathologic examination of prostate. The results have been found encouraging. The cytodiagnostic accuracy rate was 99 p.c. in identification of benign or malignant nature of the lesions and 94 p.c. in cytologic specific diagnosis (exactly similar to histopathologic diagnosis). The technique was simple, rapid, economical, reliable, easy to repeat and highly suitable for outpatients. The procedure can routinely be adopted in places where facilities for preparation of paraffin section do not exist or when quick diagnosis is required.


Plasmodium falciparum invades human red cells via a parasite produced glycosidase. This report describes that P. falciparum produces a neuraminidase like activity on invasion into erythrocytes in culture on the basis of biochemical and immunological investigations. This activity in turn modifies the surface glycoprotein receptors of red cells and may be of help in the inhibition of further invasion by merozoites. The characterization of this enzyme activity may help elucidate the mechanism of cerebral malaria.


A search for immunocontraceptive agent from marine sources--role of antispermatheca globulin of Telescopium telescopium on fertility regulation in male rat. Polyclonal antisera were developed in rabbits against 'spermatheca', the reproductive gland of T. telescopium, a marine mollusc. The gland contains spermatozoa. Antisera and its IgG fraction (ASTG) indicated common antigenic determinants by immunodiffusion and had titer values 81920 and 1280 against crude antigen extract. Cycling female rats when exposed to passively immunized male rats with different doses of ASTG, had reduction in implantation sites and litter size. Females had pseudopregnancy when exposed to higher doses of immunized males and had normal cycle after 20 days in average. ASTG in male rats caused decrease in weight of the reproductive glands, alteration in sperm concentration, motility and morphology, formation of multinucleated giant cells and vacuoles leading to arrest of spermatogenesis and reduction in seminiferous tubular diameter. The effects were dose dependent with reversible infertility. The results indicate presence of a common antigenic determinants which cross-react with vertebrates and existence of common relation through phylogenetic evolution and their immune responses.


Effect of lysine residue modification of ovine luteinizing hormone by heterobifunctional crosslinking reagent SPDP on subunit-subunit association, receptor binding and biological activity. The increasing use of heterobifunctional crosslinking agent in the design of hormone-carrier conjugates for selective targeting or inducing immune response against the hormone has prompted us to study the effect of epsilon-NH2 group modification of oLH-subunit, their recombination, immunoreactivity, receptor binding and biological activity. The epsilon-NH2 groups of oLH alpha and oLH beta subunits were modified by using SPDP. The SPDP modified oLH alpha derivatives hybridize to native OLH beta as judged by RP-HPLC analysis. The sequential modification of alpha and beta subunits led to progressive reduction in immunoreactivity and receptor binding activities. The steroidogenic potential of oLH beta.SPDP.alpha oLH recombinant was relatively comparable. The modification of six or more epsilon-NH2 groups in oLH alpha although recombine fully with native oLH beta but failed to react to anti-oLH antibody. Moreover, steroidogenic activity was also abolished. Introduction up to four SPDP groups in oLH alpha compromised immunological and biological activities but further addition of two more SPDP groups completely abolished antibody reactivity, receptor binding and steroidogenic activity indicating the importance of later two -NH2 groups in the receptor recognition and steroidogenic potential.


Termination of pregnancy in mice following antiserum administration to modified chicken riboflavin carrier protein. Rabbit antibodies to native riboflavin carrier protein (RCP), are to a large extent directed towards conformational epitopes and antibodies to disulphide bond reduced carboxymethylated RCP (RCM-RCP) are towards sequential epitopes. The major cyanogen bromide (CNBr) fragments and tryptic fragments of RCM-RCP interact with both antiserum to RCM-RCP and RCP. Passive immunization of pregnant mice with antibodies to RCM-RCP results in bioneutralization, leading to termination of pregnancy. Recently, a major tryptic fragment of RCM-RCP (24 +/- 2 kd) which could assume conformation at the antibody combining site of native RCP, obtained following mild trypsinization has been identified [Natraj et al. J. Biosci, 15 (1990) 341]. Rabbit antibodies to RCM-RCP treated with trypsin generated antibodies of low titer which interacted with RCM-RCP as well as RCP. The interaction of this antibody with RCP was of high affinity and could be displaced with RCP. The bioneutralizing ability of the antibody was demonstrated by its ability to cause termination of pregnancy in mice.


Characterization of a neutralizing monoclonal antibody to the external glycoprotein of HIV-1. The major neutralizing epitope on the external glycoprotein of HIV-1 was studied with an envelope-specific monoclonal antibody and with a human serum positive for antibodies to HIV-1 proteins, both of which were able to neutralize virus infectivity. The monoclonal antibody reacted specifically with gp120 from HIV-1IIIB, and was shown to neutralize infection of CEM cells by cell-free virions, and inhibited the formation of syncytia normally observed when uninfected cells are cocultured with HIV-1-infected cells. Similar neutralization of viral infection and inhibition of syncytia formation was also demonstrated by the HIV-1-antibody-positive human serum. By examining a number of overlapping peptides from a region of HIV-1 gp120 known to contain a neutralizing epitope, this epitope was localized between amino acids 307 and 320 (V3 loop) in the external glycoprotein molecule. The monoclonal antibody did not interfere with the binding of gp120 to CD4, or with the subsequent step of CD4-induced shedding of gp120 from the viral envelope. However, it blocked the proteolytic cleavage of the V3 loop by thrombin, suggesting that the antibody may be inhibiting the interaction of the loop with other membrane-bound proteins.


Hepatitis C virus infection in patients with acute hepatitis B. The prevalence of antibodies to hepatitis C virus (anti-HCV) was studied using a second-generation ELISA test in 121 patients with self-limiting acute hepatitis B, including 63 intravenous drug addicts (IVDA). Within the first month after the onset of illness, 47.1% of the patients were anti-HCV positive, this figure reaching 52.1% six months later. The prevalence in the sixth month was significantly higher in the IVDA (93.6%) than in the non-IVDA (6.9%) (p < 0.00001). Among the IVDA, anti-HCV was more frequent in those with (100%) than in those without hepatitis delta virus (HDV) coinfection (84.6%) (p = 0.004). Of the 63 anti-HCV positive patients, 36 (57.1%) continued to exhibit abnormal transaminase levels for more than six months, while this was not observed in anti-HCV negative patients. These results show a high prevalence of infection by hepatitis C virus (HCV) in IVDA with acute B hepatitis. As a rule, infection by HCV occurred prior to the hepatitis B infection, although occasionally simultaneous infections were observed. HCV appears to be the agent responsible for chronic liver disease in patients with acute B hepatitis who become HBsAg negative.


Close correlation between hepatitis C virus serology and polymerase chain reaction in chronically infected patients. To determine whether the persistent presence of antibodies to recombinant antigens of the hepatitis C virus (HCV) corresponds to the presence of hepatitis C virus RNA in the same serum, 85 anti-HCV positive patients were studied by the polymerase chain reaction (PCR). The focus of the research was on patients with chronic hepatitis. Eighty- three patients were found to be positive by PCR; only two were negative. In addition, liver biopsies taken from seven patients positive for anti-HCV were shown to contain HCV-specific RNA. Sera collected from three patients suspected to have NANB hepatitis on the basis of clinical symptoms were negative both for HCV antibodies and HCV RNA. The correlation between HCV antibody positivity and detection of HCV RNA was 97.6%.


Enzyme and immunohistochemical assessment of myocardial damage after ischaemia and reperfusion in a closed-chest pig model. The usefulness of different enzyme and immunohistochemical stains to distinguish reversible and irreversible myocardial cell injury after experimental coronary artery occlusion of varying duration and reperfusion with or without superoxide dismutase as adjunct was investigated. Biopsies or parts of the infarcted and non-infarcted area were rapidly frozen and sectioned in series for enzyme and immunohistochemical evaluation. Sections were stained for the demonstration of phosphorylase, myofibrillar ATPase and mitochondrial oxidative enzymes and also with periodic acid-Schiff, alizarin red S and routine histological stains. Other sections in series were stained with antibodies against fibronectin and the intermediate filament proteins desmin and vimentin. In 49 biopsies a blind quantitative estimation of the area stained for fibronectin, phosphorylase and alizarin red S was performed and evaluated statistically. Phosphorylase, periodic acid-Schiff, fibronectin and alizarin red S allowed delineation of affected myocardium after 30 min of ischaemia followed by reperfusion whereas with the other stains, affected myocardium was readily detectable only after 60 or 90 min of ischaemia followed by reperfusion as well as after 24 h of ischaemia without reperfusion. The immunostaining for fibronectin was very distinct and inversely related to the phosphorylase activity. We show that fibronectin is an excellent marker for damaged cells and that these positively stained myocytes are necrotic as confirmed ultrastructurally. Using alizarin red S as a marker of calcium accumulation in myocytes, a marked discrepancy was observed between the area of fibronectin-containing myocytes and that of myocytes stained by alizarin red S. Calcium accumulation in mitochondria is thus not a prerequisite for myocyte necrosis but does occur only in some of the irreversibly damaged cells. Of special interest is the finding that there was a significant reduction of intracellular calcium in pigs where superoxide dismutase had been used as an adjunct at reperfusion, thus supporting the theory that free radicals do play a role during reperfusion of ischaemic myocardium.


T-cell receptor beta usage by 35 different antigen-specific T-cell clones restricted by HLA-Dw4 or -Dw14.1. We studied whether antigen-specific T cells being restricted by the very similar HLA-Dw4 and/or -Dw14.1 molecules might demonstrate homogeneities in parts of their TCR. TCCs were generated from three individuals who were all HLA-Dw4/Dw14.1 heterozygous. Thirty-five TCCs specific for PPD or TT and restricted by HLA-Dw4 and/or -Dw14.1 were selected for TCR beta gene sequencing. We found that 19 different V beta genes from 13 V beta families were expressed by these TCCs. Thus, it seems that many different TCRV beta genes may be used by TCCs restricted by these HLA molecules. For PPD-specific TCCs, a possible biased usage of V beta 8, as well as possible preferential usage of a CDR3 motif, were found.


Cytomegalovirus- and interferon-related effects on human endothelial cells. Cytomegalovirus infection reduces upregulation of HLA class II antigen expression after treatment with interferon-gamma. Cultured human umbilical vein endothelial cells (HUVEs) were infected with human cytomegalovirus (HCMV) strain AD169. Up to 50% HUVEs proved to be positive for HCMV early nuclear antigens 24 hours after inoculation with virus. Following infection kinetics of surface expression of HLA class I and II, intercellular adhesion molecule (ICAM-1) and endothelial lymphocyte adhesion molecule (ELAM-1) on HUVEs were investigated by means of flow cytometry. A slight increase in HLA class I expression was observed, whereas expression of HLA class II (DR, DP, DQ) antigens was not induced by infection with HCMV. Furthermore, when compared with uninfected cells treated with interferon-gamma (IFN-gamma), reduced enhancement of HLA-DR expression was conspicuous in HCMV-infected cells treated with IFN-gamma. There is evidence that only a portion of HUVE is affected in its ability to upregulate HLA class II antigens. While expression of ICAM-1 was found to be enhanced between 8 and 20 hours after infection with a maximum at 12 hours after infection, no modulation of ELAM-1 was seen.


Sperm surface proteins after capacitation. Expression of vitronectin on the spermatozoan head and laminin on the sperm tail. Several integrins recognize as ligands proteins containing the Arg-Gly-Asp (RGD) sequence, such as fibronectin, vitronectin, and laminin. It has been previously demonstrated that oligopeptides containing the RGD sequence competitively inhibit both the adhesion of hamster and human sperm to zona-free hamster eggs and their subsequent penetration. In addition, the appearance of fibronectin on the surface of living human spermatozoa after capacitation has been demonstrated. In this work, it is shown that spermatozoa incubated overnight under capacitating conditions, but not fresh spermatozoa, also display the RGD-containing proteins vitronectin and laminin. Whereas the expression of fibronectin does not appear to be localized to any specific region of the sperm surface, laminin is present solely on the sperm tail, and vitronectin was detected mostly as an equatorial band on the sperm head. The percent of capacitated spermatozoa within each ejaculate reacting with antivitronectin antibodies (51% to 94%) was similar to that observed with antifibronectin antibodies (72% to 100%) in a series of fertile donors, and in a series of infertile men (7% to 98% for vitronectin versus 5% to 100% for fibronectin). In contrast, the percent of spermatozoa displaying laminin was lower, ranging from 2% to 42% for fertile donors and from 5% to 34% for infertile donors, and was unrelated to the expression of fibronectin or vitronectin. The time of appearance of both fibronectin and vitronectin when spermatozoa were incubated under capacitating conditions varied for different sperm donors, suggesting a difference in the process of their expression between different men. The specificity of antivitronectin antibody binding to human spermatozoa was demonstrated by competitive inhibition with purified human vitronectin. That there was no immunologic reaction between the antivitronectin antibodies used and fibronectin was demonstrated both by the failure of free fibronectin to inhibit antivitronectin antibody binding to spermatozoa, and by Dot blot analysis. A partial cross-reaction of the polyclonal antivitronectin antibody with fibronectin was shown by Western blot analysis, but this phenomenon was not present when the monoclonal antivitronectin antibody was used. In addition, both fibronectin and vitronectin could be extracted from capacitated spermatozoa solubilized in Chaps buffer, as shown by Dot blot and Western blot analysis. These observations suggest that vitronectin and fibronectin expressed on the surface of capacitated human spermatozoa could act as a ligand for specific receptors on the egg, and play a role in sperm-oolemmal adhesion.


Proteins of the cornified envelope.

Proteins of the cornified envelope. The cornified envelope of keratinocytes is an insoluble structure formed beneath the plasma membrane at the base of the stratum corneum. It is made by cross-linking precursor proteins by a membrane-associated transglutaminase. Using the cornified envelope of cultured human keratinocytes as the immunogen, we obtained a number of monoclonal antibodies which stained epidermis in a variety of ways. The peripheral staining pattern has been associated with several envelope precursors and this has been confirmed by western blots. A mouse IgM monoclonal antibody directed against epidermal basal cell hemidesmosomes was also discovered. By immunofluorescence, the monoclonal antibody produced a strong linear staining of the basement membrane zone and a polar cap on trypsin-dissociated epidermal basal cells. By immunoelectron microscopy, immunoreactants were present in the attachment plaques of hemidesmosomes on guinea pig esophagus. However, no protein reactive with the antibody was detected. This study suggests that an antigen associated with the basal cell hemidesmosomes may be incorporated in the cornified envelope.


Keratohyalin, trichohyalin and keratohyalin-trichohyalin hybrid granules: an overview. Filaggrin and trichohyalin are the two major intermediate filament associated proteins which interact with keratin filaments in the skin. These two proteins initially accumulate in cytoplasmic granules called keratohyalin or trichohyalin granules which provide prominent morphological hallmarks of differentiation in the epidermis and the inner root sheath of hair follicles, respectively. The contents of each of these granules are modified and subsequently released into the cytoplasm of the fully mature cells where they function in the role of aggregating keratin filament bundles. We are beginning to identify several important aspects relative to the unique biological functions of both filaggrin and trichohyalin during the late stages of keratinocyte differentiation. This overview summarizes recent work on these proteins and will also highlight the existence of novel cytoplasmic granules, keratohyalin-trichohyalin hybrid granules, in dorsal tongue epithelia.


Control of the distribution of hemidesmosome components in cultured keratinocytes: Ca2+ and phorbol esters. DJM-1 cells (a human squamous cell carcinoma cell line) grown in low Ca2+ medium did not form cell-cell junctions of desmosome-keratin intermediate filament (KIF). When they were shifted to normal (high) Ca2+ medium, rapid translocation of desmoplakins from the cytosol to the plasma membrane to form desmosomes and reorganization of 180 kd-hemidesmosome proteins were induced almost simultaneously. In correlation with these morphological responses, the Ca2+ shift caused a breakdown of inositol phospholipids, a formation of diacylglycerol (DAG) and inositol trisphosphate (IP3), protein kinase C (PKC) activation, and Ca2+ influx. 12-0-tetradecanoylphorbol-13-acetate (TPA)-treatment of low Ca(2+)-grown DJM-1 cells also caused desmosome formation in association with PKC activation. These TPA effects were cancelled with PKC inhibitors, 1-(5-isoquinolinylsulfomyl)-2-methylpiperazine (H7) and staurosporine. Treatment with other PKC-activating agents, phorbol-12,13-butyrate (PDBu) and diaoctanoylglycerol (DOG), also induced desmosome formation. TPA-treatment of normal Ca(2+)-grown cells collapsed the organized distribution of the 180 kd-hemidesmosome protein and appeared to detach this protein from the cell-matrix adhering sites. This effect was also inhibited by H7. These results suggest that PKC activation plays important roles in upregulation of cell-cell junctions and downregulation of cell-matrix junctions in association with differentiation of keratinocytes.


Regulation of epidermal keratin expression by retinoic acid and thyroid hormone. In the epidermis, retinoic acid (RA) is known to regulate the gene expression of keratins, the intermediate filament proteins of epithelial cells. We have cloned the upstream regulatory regions of three human epidermal keratin genes, K5, K10, and K14, and engineered DNA constructs in which these regions drive expression of the CAT reporter gene. By co-transfecting the constructs into various epithelial cell types along with the vectors expressing the nuclear receptors for RA and thyroid hormone (T3), we have shown that RA and T3 directly regulate expression of these three keratin genes through the action of their nuclear receptors. In this paper, we review our previous results to stress that RA has a dual effect on keratin expression in epidermis: both direct and indirect. We also analyze the DNA sequences upstream from those three RA-regulated keratin genes and identify the clusters of degenerate consensus half-site motifs, which may comprise the putative retinoic acid recognition elements (RAREs). Furthermore, our recent results concerning the regulation of K5 and K14 expression by the RA receptor are also shown; these confirm our predictions regarding the location of the RAREs in epidermal keratin genes.


Evidence of differentiated keratin peptide (K1) in cultured human squamous cell carcinomas: demonstration of generality by three different approaches. The largest keratin peptide (K1, 68KD) has not been detectable in cultured human squamous cell carcinomas. However, quite recently, the K1 peptide was clarified to be present in two kinds of cultured HSC by using a low salt aqueous solution, rather than the high salt and Triton X-100 employed by many previous researchers (Biochem. Biophys. Res. Commun., 182, 1440-1445, 1992). To determine whether this phenomenon is common or not in cultured HSCs, I further demonstrated the K1 peptide by extracting it with two different buffers and by 2D-PAGE, immunological techniques, and Northern blot analysis, using another kind of HSC. Until now, keratin extraction has been done using high salt/Triton X-100 solution, during which K1 peptide may be removed because it has developed an affinity with the buffer. Many investigators may have therefore overlooked it.


Inactivation of keratin genes by gene targeting: a perspective. Recent technological advances in experimental molecular genetics have provided an opportunity for determining the function of any gene including those which are important in different epithelia. This report summarizes some recent results of experiments utilizing gene targeting technology to determine the function of intermediate filaments and emphasizes both the problems and the potential of applying this technology to problems in dermatology.


In vitro keratin expression of hair cells. Human hair follicles were isolated from the scalp by dispase and collagenase treatment and dispersed into a cell suspension by trypsin. These cells proliferated well and could be subcultured 7 to 8 times. The medium used was MCDB 153 HAA medium further supplemented with some amino acids, hydrocortisone, insulin, EGF, and bovine brain extract. The concentration of Ca++ was adjusted to 0.1 mM. Immunohistochemically, these cells were proved to possess keratins specific to hair forming cells.


Dysregulated expression of cellular adhesion molecules in autoimmune-prone mice. The expression of various cell adhesion molecules on lymphocytes and endothelial cells was examined. The strong expression of CD44 antigen on abnormal double negative T cells of lymph nodes was characteristic in MRL-lpr mice. The accumulation of abnormal double negative T cells in swelling lymph nodes was also predominant in MRL-lpr mice; this may result from augmented binding of lymphocytes to endothelial cell surfaces of lymph nodes, possibly due to the failure of Mel-14 antigen shedding from abnormal lymphocyte cell surface. The expression of VCAM-1 antigen was detected in endothelial cell surface of lymph nodes in MRL-lpr mice, which also explains the accumulation of lymphocytes in lymph nodes. However, the expression of VLA-4 antigen on lymphocytes which interact with VCAM-1 on endothelial cells was not significantly different between MRL-lpr and MRL(-)+ mice.


Analysis of expression and soluble form of intercellular adhesion molecule-1 in malignant melanoma. The effects of interferon (IFN) on the expression of ICAM-1 in human melanoma cell lines and the shedding of ICAM-1 into spent media were investigated using an indirect binding assay and a double determinant immunoassay (DDIA). While IFN increased the level of expression of ICAM-1 on melanoma cell lines, the susceptibility to IFN differed according to cell line. The enhancing effect of IFN-gamma was concentration-dependent, and it exceeded those of IFN-alpha and IFN-beta. Shedding of ICAM-1 into spent media was detected effectively, and the results agreed well with the expression of ICAM-1 on melanoma cells. Immunostaining of the surgically removed melanoma lesions was positively correlated with the thickness of the primary lesion. The expression of ICAM-1 in primary melanoma lesions was inversely correlated with the disease-free interval. The circulating ICAM-1 with stage 4 melanoma patients significantly exceeded that of stage 1-3 melanoma patients. These observations suggest that ICAM-1 in its soluble form may play an important role in host immunities and may be useful in evaluating the prognosis of patients with melanoma.


Transitory elevation of serum amylase levels after restorative proctocolectomy. The incidence of postoperative hyperamylasaemia was evaluated in 70 patients who underwent staged restorative proctocolectomy. On the 14th postoperative day, 27 of 70 patients after total colectomy showed hyperamylasaemia, and the serum amylase returned to normal on the 30th postoperative day. Three out of 37 after ileoanal anastomosis and 8 out of 70 after stoma closure showed elevation of serum amylase postoperatively. Total colectomy might have a possible role in the postoperative increase of serum amylase. None of these patients had any obvious clinical evidence of postoperative pancreatitis. In 7 patients showing significant elevation of serum amylase levels (over 1000 IU), this was due predominantly to the pancreatic isoenzyme. This transitory elevation of serum amylase did not seem to be altered by the administration of therapeutic agents for pancreatitis. These observations suggest that postoperative hyperamylasaemia without clinical evidence of pancreatitis is very common after total colectomy, and that postoperative hyperamylasaemia itself does not necessarily require treatment.


Ionic mechanisms of transient inward current in the absence of Na(+)-Ca2+ exchange in rabbit cardiac Purkinje fibres. 1. Membrane currents were measured with a two-microelectrode technique in voltage clamped rabbit cardiac Purkinje fibres under conditions known to cause intracellular calcium overload and to eliminate or minimize Na(+)-Ca2+ exchange. 2. Increasing [Ca2+]o from 2.5 to 5 mM or above and substituting external sodium with either sucrose, choline or Li+ induced an oscillatory transient inward current (TI) which peaked 200-300 ms after repolarization from a previous depolarizing pulse. The TI quickly disappeared upon return to normal Tyrode solution. Both the rate and configuration of action potentials of Purkinje fibres also returned to control upon return to Tyrode solution after 30 min of high Ca2+ exposure, if the Ca2+ concentration was 30 mM or less. 3. The TI in Na(+)-free solution was Ca2+ dependent. Either zero or low (2.5 mM) [Ca2+]o, or replacement of [Ca2+]o by BaCl prevented induction of the TI current upon repolarization from a previous depolarizing pulse. 4. In the presence of 30 mM-CaCl2 and with choline chloride as the substitute for NaCl, TI had a distinct reversal potential (Erev) of -25 mV. The time-to-peak TI, either inward or outward, did not shift significantly with change in voltage. Both inward and outward TI were simultaneously abolished by exposure to 1 microM-ryanodine, suggesting they were both activated by transient release of Ca2+ from the sarcoplasmic reticulum. The occurrence of TI in the absence of [Na+]o is not compatible with an electrogenic Na(+)-Ca2+ exchange mechanism. The existence of a clear-cut reversal potential suggests that an ionic channel may be responsible for the TI under these conditions. 5. Both the magnitude of peak TI and the Erev were affected by changes of CaCl2 concentration. (i) Under steady-state conditions, peak inward TI was significantly increased when the [Ca2+]o was elevated from 5 to 15 mM. The peak TI in the outward direction was significantly increased when [Ca2+]o was elevated from 15 to 30 mM; however, the difference in peak inward TI at 15 and 30 mM [Ca2+]o was small. (ii) Clear-cut reversals of TI were found at Ca2+ concentrations of 10 mM (Erev = -19.5 mV) or greater, and elevation of [Ca2+]o to 20, 30, 50 and 105 mM shifted the Erev to more negative potentials. (iii) In the presence of 5 mM [Ca2+]o the inward TI declined to zero at about -30 mV, and test voltages between -55 and +5 mV failed to reveal a distinct outward TI.(ABSTRACT TRUNCATED AT 400 WORDS)


Stretch-activated anion currents of rabbit cardiac myocytes. 1. Stretch-activated anion currents were studied in sino-atrial and atrial cells using the whole-cell patch clamp technique. With continuous application of positive pressure (5-15 cmH2O) through the patch clamp electrode, the cell was inflated and the membrane conductance was increased. 2. Voltage clamp steps revealed that the stretch-activated currents had time-independent characteristics. The increase in membrane conductance was reversible on subsequent application of negative pressure to the electrode. 3. The reversal potential of the stretch-activated currents was shifted by 60 mV for a 10-fold change in intracellular Cl- concentration, while it was unaffected by replacement of Na+ in the extracellular solution by N-methyl-D-glucamine. Cell superfusion with Cl(-)-deficient solution (10 mM Cl-) reduced the amplitude of outward current. These findings indicate that the stretch-activated conductance is Cl- selective. 4. The sequence of anion permeability through the stretch-activated conductance was determined to be I-(1.7) > NO3-(1.5) > Br-(1.2) > Cl-(1.0) > and F-(0.6). SCN- appeared to be more permeant than I-. 5. The stretch-activated conductance was reduced by the Cl- channel blockers, 4,4'-dinitrostilbene-2,2'-disulphonic acid disodium salt, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid or anthracene-9-carboxylate (9-AC). Administration of furosemide or bumetanide had no effect. 6. The stretch-activated Cl- current was recorded even though intracellular Ca2+ ions were chelated by including 10 mM EGTA in the pipette solution. Neither the specific peptide inhibitor of cyclic AMP-dependent protein kinase (50 microM), nor the non-selective blocker of protein kinases, H-7 (20 microM), was effective in reducing the stretch-activated Cl- current, suggesting that the stretch-activated Cl- current is a novel type of cardiac Cl- current, which shows a different modulatory mechanism from that of other cardiac Cl- currents.