Definition of immunogenic determinants of the human papillomavirus type 16 nucleoprotein E7. Specific T lymphocyte lines and T cell clones were established from peripheral blood mononuclear cells of asymptomatic seropositive individuals employing synthetic peptides which correspond to the sequence of the human papillomavirus (HPV) type 16 transforming protein E7. Specificity analysis of T cells as determined by means of [3H] thymidine incorporation after stimulation with individual peptides revealed three immunogenic determinants of E7 that are recognised in association with at least two different HLA haplotypes. One N-terminal region (aminoacids 5-18) was recognised by one T cell line. T cell clones and the corresponding T cell line established from another donor responded to a different N-terminal (17-38) and to a C-terminal region (69-86). The N-terminal sequence 5-18 and the C-terminal determinant contain a periodicity of hydrophilic and hydrophobic residues that have been found in many T cell epitopes. Phenotypic characterisation of T cell clones by indirect immunofluorescence revealed that the T cell clones expressed the CD4 surface glycoprotein suggesting that the specific E7 determinants were recognised in association with major histocompatibility complex (MHC) class II molecules. With regard to functional properties, at least three T cell clones exhibited specific cytotoxic activity towards autologous B lymphocytes transformed by Epstein-Barr virus in the presence of the relevant HPV16 E7 peptides. The implications of these results regarding the development of vaccination strategies and host-virus interaction are discussed.


Aneuploid subpopulations in tumour-invaded lymph nodes from breast cancer patients. Fresh, paired primary tumours and lymph node metastases from breast cancer patients were compared by DNA flow cytometry. Although 65% of primary tumours were aneuploid, the detection of aneuploid peaks in corresponding nodal metastases was rare (only 6 cases out of 25) in single-parameter DNA analysis. Detection of aneuploid subpopulations in lymph nodes was greatly improved in dual-parameter DNA analysis using an anticytokeratin (CK) antibody which allowed ploidy determination on CK+ epithelial cells alone. Examination of 12 lymph nodes for CK+ cells revealed the presence of both diploid and aneuploid tumour cells in tumour invaded nodes. In patients with multiploid primary tumours, a subpopulation of the primary aneuploid cells was dominant in the nodal metastases. This suggests that aneuploidy is an integral property of metastatic cells and that within a primary tumour a subpopulation may have a higher metastatic potential.


Cytotoxicity of 5-aza-2'-deoxycytidine in a mammalian cell system. After the addition of 5-aza-2'-deoxycytidine, a potent inhibitor of DNA methylation and S-phase-specific cytotoxic agent, metaphase chromosomes of Chinese hamster ovary (CHO) cells exhibited a highly decondensed and extended morphology (numerous "fragile sites") at the first mitotic division. However, when a lethal dose of this drug was added in early G1 phase to cells synchronised by mitotic selection, the majority subsequently divided at the same time as an untreated control cell population with few division abnormalities and with few of the more usual types of chromosome aberrations such as gaps, breaks and exchanges. The drug-treated cells also entered and completed the second S-phase without significant delay and it was only at the second mitosis after addition of 5-azadeoxycytidine that cells showed delays in entering mitosis and significant increases in abnormal divisions concomitant with a modest increase in chromosome aberrations. If cells in a tumour behave similarly, the tumour mass would be expected to double before any reduction in tumour burden could be expected to occur.


Expression of pp60c-src in human small cell and non-small cell lung carcinomas. c-src protein was found in 60% of lung carcinomas (20 of 33 cases or primary tumours) by immunoblotting with a monoclonal antibody (Mab 327) and immunohistochemistry with serum from rabbits bearing tumours induced by Rous sarcoma virus. src protein expression was assessed in 4 small cell lung carcinomas and in an atypical carcinoid of neuroendocrine origin. However, pp60c-src was also found in non-small cell lung carcinomas: in 60-80% of adenocarcinomas and bronchiolo-alveolar cancers and in 50% of squamous cell carcinomas. In the squamous cell carcinomas, src protein was expressed more frequently in poorly differentiated than in well and moderately differentiated carcinomas. Expression of pp60c-src was not found in epithelial cells of histologically unchanged lung tissues. These results show that pp60c-src may be activated in human lung carcinomas of different histopathological types.


The capacity of peripheral blood stem cells mobilised with chemotherapy plus G-CSF to repopulate irradiated marrow stroma in vitro is similar to that of bone marrow. After treatment of patients with intermediate or high grade non-Hodgkin lymphoma with chemotherapy plus G-CSF the numbers of haemopoietic progenitor cells in the circulation increased to a mean of 226-fold for mixed CFC (Mix-CFC), 278-fold for GM-CFC and 29-fold for erythroid burst forming unit (BFU-E). The mean increase was modest (7-12-fold) for patients treated with chemotherapy alone. Peripheral blood mononuclear cells harvested at the time of the peak in the numbers of progenitors, or 2-4 days before the peak, seeded onto irradiated marrow stroma in vitro, repopulated the stroma and generated active haemopoiesis at least as effectively as bone marrow cells on a cell per cell basis. This is in contrast to the poor repopulating capacity of pretreatment blood. The results indicate that not only the progenitor cells, but also the repopulating stem cells migrated into the blood after chemotherapy plus G-CSF in sufficient numbers to allow harvesting and successful grafting without the possible complication of late haemopoietic failure.


Serum lactate dehydrogenase isoenzyme 1 and tumour volume are indicators of response to treatment and predictors of prognosis in metastatic testicular germ cell tumours. 44 patients with metastatic testicular germ cell tumours treated with cisplatin-based chemotherapy were evaluated for prognostic implications of clinical characteristics. 22 obtained complete remission by the initial chemotherapy, and 30 are disease-free. S-LDH-1 had an overall predictive value regarding the response of 80%, S-LDH of 64%, S-AFP of 62%, and S-hCG of 62%. In multivariate analysis regarding response, only tumour volume classified according to the Royal Marsden system (P = 0.0036) and S-LDH-1 (P = 0.0069) yielded information. Regarding survival, S-LDH-1 (P = 0.0141) and an estimate of total tumour mass (P = 0.0171) had most impact with additional information from S-hCG only (P = 0.0536). We conclude that S-LDH-1 may be used as a tumour marker in addition to S-hCG and S-AFP in patients with metastatic testicular germ cell tumour.


[Tendinitis of the palmaris longus muscle. Apropos of 24 cases]. Twenty-four cases of flexor carpi radialis tendinitis were diagnosed between 1985 and 1989 by SOS Main Strasbourg. This condition typically occurs in women (75%) with a mean age of 47 years with no particular occupational activity who present with spontaneous and induced pain along the distal part of the tendon. Examination may reveal disturbances of cutaneous sensation in the territory of the palmar cutaneous branch of the median nerve and associated signs: cysts (10/24 cases), scapho-trapezium osteoarthrosis (3 documented cases). Medical treatment provided good results in 7 out of 9 patients reviewed after a mean follow-up of 15 months. In the event of failure, surgical treatment with opening of the compartment, provided 4 good results in 6 patients reviewed after a mean follow-up of 2 years 4 months.


[The value of pollicization in the reconstruction of thumb injuries in adults. Observations apropos of 15 cases]. Pollicisation remains a good solution to restore the function of the adult thumb after injury. This technique requires a good knowledge of plastic hand surgery to perform an emergency or secondary reconstruction, sparing the functional integrity and enhancing the value of the stumps. The authors report their own experience of 15 cases treated between 1985-1990.


[The Sauvé-Kapandji operation. Analysis and results of 69 cases]. The authors present the results of 69 cases operated between 1983 and 1989. The indications were dominated by two aetiologies: degenerative lesions of the inferior radio-ulnar joint in rheumatoid arthritis: 58% of cases, and traumatic sequelae, predominantly secondary to fractures of the distal radius: 34% of cases. The authors stress the effects of distant lesions: skeletal lesions of the two bones of the forearm or the superior radio-ulnar joint. A third group of rarer indications consisted of painful dislocations of the inferior radio-ulnar joint in Madelung's disease (indication only applied to adults). There were two types of complications: complications due to excessively long or intra-articular screws, which were treated by removal of the screws and, more severe, ossifications of the zone of diaphyseal resection (5 cases) which interfered with the functional result in terms of mobility. Three cases required extensive diaphyseal resection. The results are analysed for each aetiological group in terms of pain, mobility prehensile strength after more than one year of follow-up. Good results were obtained in 80% of cases of post-traumatic lesions (16 cases). The insufficient results were related to associated lesions of the elbow or the 2 bones of the forearm. A good result was obtained in 3 out of 4 cases of Madelung's disease and one case, operated elsewhere and revised by pseudoarthrodesis, obtained a moderate result. Lastly, in rheumatoid arthritis, the results in terms of pain, mobility and prehensile strength were good, apart from 2 failures due to progression of rheumatoid disease (cases unsuitable for this treatment). Radiographic analysis according to Youm and Mac Murtry's criteria showed minimal regression in the carpoulnar translation index (pre-op: 0.28; post-op: 0.22). Overall, the Sauvé-Kapandji operation provided good quality clinical results when the indications and technique were rigorously applied.


[Description of a vascularized bone graft taken from the head of the 2nd metacarpal bone]. The authors describe a new vascularised bone graft from the distal part of the second metacarpal. They report an anatomic study on 20 cadavers which demonstrates the existence of two dorsal first web arteries (superficial and deep). The existence of free anastomosis between the two arteries is demonstrated. The vascularisation of the bone graft relies upon the deep first web artery which lies against the lateral aspect of the bone. The operative procedure is described and the authors stress the necessity to transfer the vascularized bone graft with a large soft tissue pedicle.


[The carpal bump].

[The carpal bump]. Fiolle reported the first description of a "carpal boss". The "hump" is cosmetically disturbing and sometimes painful. It has to be distinguished from a ganglion. A lateral X-Ray with 30 degrees of supination reveals a spur or a styloid bone (12.5%). In a series of 44 patients, treated over a period of 7 years, 21 needed operation for pain or a complication of previous surgery. The technique described by Cuono and Watson was successfully used without recurrence of deformity but in 2 cases some pain remained during strenuous efforts.


[Chronic ulnar nerve compression syndrome at the elbow. Apropos of 74 cases]. Seventy-four patients were operated at Bichat hospital for chronic ulnar nerve entrapment at the elbow between 1982 and 1988. For 62 of them, the etiology of the compression was idiopathic and these cases were treated by neurolysis only or, if the nerve was unstable, by neurolysis associated with medial epicondylectomy. For 12 of them, the etiology of the compression was post-traumatic and these cases were treated by anterior subcutaneous transposition of the nerve using a fat sling. The average follow-up is 28 months and the results take into account the clinical preoperative grading according Mac Gowan's classification: grade I subjective symptoms combined with hypoesthesia in ulnar fingers grade II: weakness and wasting of the interossei combined with subjective symptoms, grade III: marked weakness and wasting of the interossei, adductor pollicis, and hypothenar muscles combined with anesthesia in ulnar fingers. The 62 idiopathic compressions treated by neurolysis only or, if the nerve was unstable, by neurolysis associated with medial epicondylectomy showed 51 very good and good results. The 12 post-traumatic compressions treated by anterior subcutaneous transposition of the nerve using a fat sling showed 9 very good and good results. The authors stress the importance of their approach which takes account of the clinical preoperative grading and the etiology of the compression in order to apply correct surgical treatment.


[Lipofibroma of the median nerve. Apropos of a case]. Lipofibroma is a rare, benign nerve tumour corresponding to diffuse fibroadipose infiltration of the nerve, dissociating the fasciculi without invading them. The authors report a case of lipofibroma of the median nerve in a 32 year old man presenting with a soft swelling of the palmar surface of the thumb. Treatment consisted of intraneurodissection of the tumour arising exclusively from the medial collateral nerve of the thumb. With a follow-up of two years, there has been no recurrence of the tumour, but the patient has persistent decreased sensation of the ulnar half of the thumb pulp. The features of the lipofibroma and the therapeutic options are discussed in the light of the data reported in the literature.


[Metastatic tumors of the soft tissues of the hand. Apropos of a case. Review of the literature]. Soft tissue metastases of the hands are rare tumours, representing 0.1% of all metastases. The authors report a personal case and review the literature concerning another 21 cases. The primary tumour is generally a lung or breast cancer. These metastases appear to be more frequent in men than in women. Their rarity can be explained by the absence of routine investigation of such sites. The development of such metastases always indicates a poor prognosis.


[Severe and inveterate internal scapho-trapezial ligament sprain. Apropos of an unusual case]. The authors report the first case in the literature of a chronic injury of the medial scaphotrapezial ligament. The main clinical feature was elective pain over the lateral margin of the carpal tunnel which was increased with effort. The pain was disabling and complete remission is obtained after surgical repair of the ligament.


[Radio-carpal amputation and prosthesis]. Radio-carpal amputation was rejected for a long time because of the difficulties of fitting both myoelectrical and mechanical prostheses, only allowing a purely aesthetic prosthesis. As a result of miniaturization of control systems and the progress in computers, we believe that this now constitutes the best site for amputation whenever it is possible. Preservation of the antero-posterior bony contours of the radius allows the use of short sockets attached by a system of clips. In this way, flexion-extension of the elbow is left free and the movements of pronation and supination can be used. Liberation of the proximal joints facilitates integration of the prosthetic hand.


[Sensory retraining of the hand using gnosis rings]. Sensory reeducation is very important. Many methods are used but it is difficult to assess their efficiency. Besides the routine methods we are using the gnostic rings which have on their periphery 5 symbols: a letter = A, a number = 3, a geometrical figure = a circle, a rough surface and a smooth convex surface. The symbols of the rings are in 3 different sizes (1.5 - 1 and 0.5 cm). The gnostic rings have 3 advantages: 1) they allow a precise evaluation in percentage. 2) they require an important brain effort, 3) they may be kept in the pocket and used several times during the day.


Quantitative and qualitative differences in IL-2 and IL-4 expression in primary and secondary T cell stimulation. Lymphocytes were concanavalin A (Con A) primed and the signal was withdrawn 4-48 h post-stimulation by alpha-methyl-D-mannopyranoside (alpha MM) treatment. Upon restimulation IL-2 and IL-4 RNA expression was found to be greatly enhanced. Re-expression of lymphokine RNA was dependent on signals delivered by Con A, anti-CD3 antibodies or phorbolester plus ionomycin, and could not be achieved by either IL-2, phorbolester or ionomycin alone. Increased IL-2 re-expression was only possible when alpha MM was added early after primary stimulation, while the ability for enhanced IL-4 RNA re-expression persisted. IL-2 and IL-4 RNA re-expression was characterized by increases in steady state precursor RNA levels and thus, presumably, increased rates of transcription. However, the high accumulation of IL-2 RNA observed upon restimulation was also due to greatly increased RNA stability (greater than 4 h versus 30 min after primary stimulation). Thus, secondary expression of IL-4 RNA is persistent and mostly due to quantitative changes in transcription, whereas enhanced re-expression of IL-2 RNA also results from altered post-transcriptional regulation. This phenotype, however, is only short lived.


Different effects of pemphigus antibody and plasmin on the distribution of keratin intermediate filaments and desmoplakins between cultured oral and epidermal keratinocytes. In order to clarify the molecular mechanism of blister formation in oral mucosa in pemphigus vulgaris (PV) comparing with that in epidermis, we analyzed the effects of PV serum on the distribution of keratin intermediate filaments (KIFs) and desmoplakins in oral as well as epidermal cultured keratinocytes by immunofluorescence microscopy using anti-keratin and anti-desmoplakin I/II monoclonal antibodies. After incubation with PV serum for 96 h at 37 degrees C, clusters of anti-keratin positive dots were formed around the nucleus in some of the keratinocytes from normal gingiva and soft palate but not in keratinocytes from tongue and skin, and desmoplakins also changed their distribution from linear arrangement at cell-cell contacts to clusters of dots around the nucleus in gingiva but not in epidermal keratinocytes. The dotted structures similar to those induced by pemphigus serum were formed also by incubation with human plasmin in gingival keratinocytes. However, no dot-formation of keratins was induced in these cells after incubation with trypsin. Furthermore, in epidermal keratinocytes, no keratin-dot formation was observed even after incubation with plasmin or trypsin. These results suggest that the dotted structures of KIFs caused by PV serum and plasmin might be a feature characteristic for the response of oral keratinocytes to PV serum and that there are some distinct differences in susceptibility to, and mode of, bulla formation between oral epithelium and epidermis.


Prostacyclin analogues inhibit tissue factor expression in the human monocytic cell line THP-1 via a cyclic AMP-dependent mechanism. Increased expression of tissue factor procoagulant by peripheral blood monocytes has been implicated in a number of thrombotic disorders. The present studies were undertaken to determine whether stable analogues of prostacyclin, a potent endothelium-derived platelet inhibitor and vasodilator, could inhibit tissue factor expression by human monocytic cells. Exposure of monocytic tumor THP-1 cells to 100 ng/ml endotoxin, 2 units/ml interleukin-1 beta, or 5 ng/ml tumor necrosis factor-alpha for 4 hours led to increased tissue factor procoagulant activity. Preincubation for 30 minutes with iloprost, ciprostene, and carbacyclin led to a dose-dependent inhibition of tissue factor expression induced by all three challenging agents. Iloprost was the most potent: 50% inhibition occurred at 5 nM, a concentration close to the reported dissociation constant for iloprost binding to the platelet prostacyclin receptor. An orally active analogue, cicaprost, was equally effective against endotoxin-induced tissue factor expression. Carbacyclin and ciprostene were 100 times less potent. Iloprost prevented the endotoxin-induced expression of tissue factor antigen on the surface of THP-1 cells, as determined by flow cytometry. Iloprost (500 pM-50 nM) increased intracellular levels of cyclic AMP. This effect was potentiated by isobutylmethylxanthine, an inhibitor of phosphodiesterase. The inhibitory effects of iloprost on tissue factor expression were also potentiated by isobutylmethylxanthine and mimicked by forskolin and dibutyryl cyclic AMP but not dibutyryl cyclic GMP. These results suggest that prostacyclin may play a role in downregulating tissue factor expression in monocytes, at least in part via elevation of intracellular levels of cyclic AMP.


Antifibrinolytic activities of alpha-N-acetyl-L-lysine methyl ester, epsilon-aminocaproic acid, and tranexamic acid. Importance of kringle interactions and active site inhibition. alpha-N-acetyl-L-lysine methyl ester (NALME) is a lysine analogue that reportedly binds to low-affinity lysine binding sites in plasmin(ogen) and miniplasmin(ogen). In the studies presented here, we show that NALME has antifibrinolytic activity; however, unlike the therapeutic agents epsilon-amino-n-caproic acid (epsilon ACA) and tranexamic acid (TEA), the activity of NALME is based on inhibition of the plasmin active site. NALME (0.1-10 mM) significantly inhibited the amidase activity of plasmin, miniplasmin, and streptokinase-plasmin complex without affecting alpha-thrombin or tissue plasminogen activator. epsilon ACA and TEA (0.1-10 mM) did not affect the amidase activity of plasmin or miniplasmin. A kinetic analysis showed that NALME is a competitive inhibitor of D-Val-L-Lys-p-nitroanilide HCl (S-2251) hydrolysis by plasmin; NALME binding to plasmin completely prevented S-2251 binding. The Kl for the plasmin-NALME interaction was 0.4 mM. epsilon ACA and TEA inhibited fibrin monomer digestion by plasmin and miniplasmin without binding to the active site of either enzyme. This result suggests that epsilon ACA and TEA function as antifibrinolytics by disrupting the noncovalent association of fibrin monomer with a domain common to both plasmin and miniplasmin (probably kringle 5). NALME inhibited fibrin monomer digestion principally by decreasing amidase activity. NALME was the only lysine analogue that prevented fragment X formation; TEA and epsilon ACA primarily inhibited the formation of fragments Y and D. When plasmin was incubated simultaneously with alpha 2-antiplasmin and alpha 2-macroglobulin, epsilon ACA increased the fraction of plasmin reacting with alpha 2-macroglobulin; NALME had no effect on the plasmin distribution.(ABSTRACT TRUNCATED AT 250 WORDS)


Pentraxin family of proteins interact specifically with phosphorylcholine and/or phosphorylethanolamine. Pentraxins are a family of serum proteins characterized by five identical subunits that are noncovalently linked. The two major types of pentraxins are C-reactive protein (CRP) and serum amyloid P component (SAP). CRP proteins are identified by their calcium-dependent interaction with phosphorylcholine. This study showed that SAP also bound to phosphorylated compounds but had a high specificity for phosphorylethanolamine. Thus, human CRP and SAP show high specificity that is complementary for the related compounds, phosphorylcholine and phosphorylethanolamine, respectively. This relationship suggests a complementary and/or related function for the pentraxins. Pentraxins from other species were also examined. Mouse SAP showed binding interactions and specificity similar to human SAP. Female protein (FP) from hamster and rat CRP showed a hybrid specificity and bound to both phosphorylethanolamine and phosphorylcholine. All of the proteins that bound phosphorylethanolamine also associated with human C4b-binding protein (C4BP). With the exception of human and rat CRP, all the proteins also bound to vesicles containing acidic phospholipids. All of these binding interactions were calcium-dependent and mutually exclusive, suggesting that they involved the same site on the protein. These findings suggest possible ways to examine the function of the pentraxins.


Comparison of N-glycosides of fetuins from different species and human alpha 2-HS-glycoprotein. Complex type N-glycosides of commercial bovine fetuin preparations from pooled fetal calf serum have been shown to contain comparable amounts of Gal4,4,4TRI (see structure A below) and Gal4,4,3TRI (structure B) as major asialo-structures. To investigate whether there is a clear genetic specificity for synthesis of these oligosaccharides, N-glycosides from two preparations of bovine fetuin, each from a single calf, were examined. Both of these structures were present in each calf, and there was only a subtle quantitative difference in the ratio of these two structures between the calves. Thus, a specific galactosyltransferase, presumably required for the biosynthesis of the Gal4,4,3TRI structure, may exist in both of these individual calves. Comparison of fetuin N-glycosides was also extended to sheep, pig, and human alpha 2-HS-glycoprotein, the human counterpart of bovine fetuin, using high-pH anion-exchange chromatography of the reducing oligosaccharides as well as HPLC of their pyridinylamino derivatives. The N-glycosides of ovine fetuin also have both Gal4,4,4TRI and Gal4,4,3TRI structures in a ratio similar to that of bovine fetuin. However, the major N-glycoside of porcine fetuin is of a fucosyl biantennary complex type structure (structure C below) and human alpha 2-HS-glycoprotein has an N-glycoside which is almost exclusively a nonfucosylated biantennary structure (structure D). This species-specific presence of N-glycosides of fetuins and comparison with N-glycosides of other glycoproteins suggest that the polypeptide sequence of a glycoprotein may affect its N-glycan structure by regulating the activity of specific glycosyltransferases. [formula: see text]


Discordant regulation of human type I collagen genes by prostaglandin E2. We examined the expression of type I collagen genes in prostaglandin E2 (PGE2)-treated human lung fibroblast cultures. Addition of PGE2 to fibroblast cultures inhibited alpha 1(I) mRNA levels by approx. 25% after 6 h and 60% after 24 h. Further studies showed that dibutyryl cAMP did not inhibit alpha 1(I) mRNA levels and that cycloheximide blocked the inhibitory effect of PGE2. In contrast, PGE2 treatment with or without cycloheximide did not affect alpha 2(I) mRNA levels. Moreover, in vitro translation of RNA derived from untreated and PGE2-treated cells yielded similar amounts of alpha 2(I) collagen peptides. Taken together, these results suggest that PGE2 induces a protein which inhibits alpha 1(I) transcription through distinct regulatory elements not under the control of cAMP and provide further evidence that the type I collagen genes can be discordantly regulated.


The increased K+ leak of malaria-infected erythrocytes is not via a Ca(2+)-activated K+ channel. Charybdotoxin and nitrendipine both inhibited K+(86Rb+) influx via the Ca(2+)-activated channel of uninfected erythrocytes but had no effect on K+(86Rb+) transport in malaria-infected cells. Activation of the channel in uninfected cells in which the cytoplasmic [Na+]/[K+] ratio was adjusted to be comparable with that of late-stage malaria-infected erythrocytes resulted in a large (nitrendipine-sensitive) increase in K+(86Rb+) influx. These results suggest that the endogenous Ca(2+)-activated K+ channel remains inactive in human red cells infected with late-stage parasites. The identity of the pathway which mediates the increased K(+)-leak in infected erythrocytes remains to be established.


Invasion and metastasis.

Invasion and metastasis. Tumor metastasis remains a major cause of death for cancer patients. From a clinical perspective, treatment of metastatic disease remains difficult. The initial tumor cell invasive and locomotive events have been completed in many patients by the time of cancer diagnosis and surgery. This observation focuses attention on the last steps in the metastatic cascade for therapeutic development, ie, angiogenesis and colonization at the metastatic site. Emerging themes in metastasis research, including the molecular characterization of protease production and angiogenesis, the interrelationship of growth and metastasis, and the genetic control of the metastatic process are discussed.


Expression of a glyceraldehyde 3-phosphate dehydrogenase gene specific to mouse spermatogenic cells. A cDNA clone encoding a putative glyceraldehyde 3-phosphate dehydrogenase (GAPD-S) protein specific to spermatogenic cells was isolated from a mouse spermatogenic cell expression library. The Gapd-s cDNA contained 1451 bp of transcribed sequence, including an ATG initiation codon and a poly(A) addition signal. The location of the Gapd-s initiation codon differed from that of other Gapd sequences, resulting in a germ cell GAPD-S protein predicted to contain 105 additional residues at the amino terminus. While GAPD is constitutively expressed in somatic tissues, Northern blot analysis demonstrated that a Gapd-s probe hybridized to a 1.5-kb transcript present only in the testis. The Gapd-s mRNA was first detected during postnatal development in the testes of 20-day-old mice, suggesting that gene expression begins shortly after the appearance of haploid round spermatids. Northern analysis of RNA from isolated mouse pachytene spermatocytes and spermatids confirmed that Gapd-s expression is confined to post-meiotic germ cells. GAPD has been previously proposed to be the key enzyme regulating glycolysis in isolated round spermatids. We hypothesize that the GAPD-S enzyme plays an important role in regulating the switch between different pathways for energy production during spermiogenesis and in the spermatozoon.


Prostaglandin synthesis regulation in human amnion tissue: involvement of protein kinase C and dependence on ribonucleic acid and protein synthesis. The role of protein kinase C (PKC) in the control of prostaglandin production by the human amnion was studied. Amnion membranes delivered spontaneously at term were minced and treated with phorbol esters, protein kinase inhibitors, cycloheximide, and actinomycin D; prostaglandin E2 (PGE2) output then was determined. Untreated tissue produced 3.97 +/- 1.13 ng PGE2/micrograms DNA/14 h (mean +/- SEM, n = 19). Phorbol dibutyrate and 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated PGE2 output up to 20-fold in a concentration-dependent manner with potencies corresponding to their efficacy as PKC activators. Four-beta-phorbol and 4-methoxy-TPA, which do not stimulate PKC, did not affect PGE2 output. Stimulation by TPA was blocked by staurosporine (IC50 = 57 nM) and H7; however, these PKC inhibitors did not decrease basal prostaglandin production. Cycloheximide inhibited basal and TPA-promoted PGE2 production and amino acid incorporation. Actinomycin D abolished TPA stimulation without decreasing unstimulated prostaglandin synthesis. These results show that amnion PGE2 production after labor is not maintained by PKC action, but PKC activation in this tissue causes a protein synthesis-dependent and RNA synthesis-dependent increase of PGE2 output. However, basal PGE2 production is dependent upon protein synthesis which, presumably, utilizes pre-existing mRNAs.


Fluid transport in a cultured cell model of kidney epithelial cyst enlargement. Madin-Darby canine kidney (MDCK) cells, when seeded into collagen gel, from polarized, spherical, epithelial cysts, which grow by a process involving fluid secretion and cell proliferation. These cysts are a useful model for understanding the dynamics of cyst enlargement in renal cystic disease. The hypothesis that MDCK cyst fluid secretion depends upon chloride secretion was tested, and a cell model for this process is presented here. Lumen and epithelial cell volumes were measured by video microscopy in acute experiments. Fluid absorption (-0.073 +/- 0.007 microliters.h-1.cm-2; N = 8) was observed when cysts were superfused with unsupplemented Dublecco's modified Eagle's medium at 36 to 37 degrees C. Fluid secretion (0.221 +/- 0.0016 microliters.h-1.cm-2; N = 25) was seen when 1 mM dibutyryl cAMP plus 0.1 mM 3-isobutyl-1-methylxanthine were added to the superfusate. cAMP-induced fluid secretion was significantly inhibited by basolateral 1 mM ouabain, 0.1 mM furosemide, or 1 mM amiloride. It was not significantly affected by 1 mM chlorothiazide, 0.01 mM bumetanide, or 0.1 mM acetazolamide in the presence of normal bicarbonate/CO2. In the nominal absence of bicarbonate/CO2 fluid secretion was 18% of control. Vasopressin-induced fluid secretion was significantly inhibited by pretreatment of cysts with 0.1 mM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Cyst cell shrinkage in isosmotic chloride-free Ringer's solution (chloride replaced by gluconate) was inhibited by 0.1 mM basolateral DIDS. The results suggest that chloride-bicarbonate exchange in the basolateral membrane of MDCK cyst epithelial cells plays a critical role in cyst fluid secretion.


Native epitopes of human IgA.

Native epitopes of human IgA. We have studied the epitopes of the human IgA molecule by using 27 monoclonal antibodies in two simple gel diffusion methods. By testing pairs of monoclonal antibodies for coprecipitation of an IgA myeloma protein, we have clearly identified sterically independent epitopes on the molecule. By testing the non-coprecipitating monoclonal antibodies with soluble IgA-monoclonal antibody immune complexes, we have identified the overlapping epitopes. Eleven proto-epitopes were mapped on the IgA molecule. Since we have not used solid-phase methods, we have presumably identified native epitopes of the IgA molecule.


Antigen-independent pathways of T-cell activation are functional in human immature thymocytes. The signal requirements for proliferation of CD1+CD3- immature thymocytes have been studied in order to define whether this immature cell population could function despite the lack of the CD3/T-cell receptor complex. We found that CD1+CD3- cells proliferate upon stimulation with anti-CD28 monoclonal antibody as well as with a pair of anti-CD2 monoclonal antibodies in the presence of low doses (0.5 ng/ml) of phorbol-13-myristate-12-acetate and/or recombinant interleukin-2. A minor fraction of CD3+ cells (15%-20%) was also present in the proliferating cell population originating from CD1+CD3- thymocytes stimulated with phorbol-13-myristate-12-acetate and recombinant interleukin-2, either in the presence or in the absence of specific monoclonal antibodies. We further observed that the anti-CD3 monoclonal antibody did not induce the proliferation of CD1+CD3- cells, as expected, and efficiently triggered unfractionated or CD1+CD3+ thymocytes only if exogenous recombinant interleukin-2 was provided. Unexpectedly, we noted that highly purified (greater than 99%), CD1+CD3- immature thymocytes could mobilize calcium via CD3, besides CD2 and CD28 surface molecules, suggesting that a minor undetectable fraction (less than 1%) of CD3+ cells was still present in the purified CD3- population. Nevertheless, the preferential expansion of CD3-CD8+ cells (about one-third of proliferating cells) after triggering via CD28, and to a lesser extent via CD2, support the notion that the alternative pathways of T-cell activation are actually functional in CD1+CD3- immature thymocytes.


A combined pseudoreplica-immunochemical technique for research and diagnostic virology. Pseudoreplica and immunochemical techniques were combined in a single protocol for identification of virus in research and clinical specimens. Stock preparations of vesicular stomatitis virus (VSV), cytomegalovirus (CMV), and herpes simplex virus (HSV) were used to develop the technique. Traditional pseudoreplicas of viral stock solutions were prepared but not negatively stained. The virus was then immunolabeled in two stages. Virus-specific polyclonal antisera were used in the first stage; colloidal gold conjugated antibodies were used as indicator antibody in the second stage. After immunolabeling, the grids were negatively stained. As a demonstration of the clinical usefulness of this approach, it was employed to antenatally identify human parvovirus B19 particles in ascites from a 22 week gestational fetus with nonimmune hydrops fetalis. The combined pseudoreplica-immunochemical approach offers several advantages over both the pseudoreplica and immunochemical methods when used in isolation. Advantages include relative purification of samples, concentration of virus, morphological preservation, and enhanced diagnostic specificity.


Section staining for electron microscopy using tannic acid as a mordant: a simple method for visualization of glycogen and collagen. The possibility that tannic acid and other classical mordants can be used in the electron microscopical section staining technique has been tested. A mordant can be defined as a chemical that combines with both a certain specific tissue component and the staining substance and thereby permits a staining reaction that otherwise will not be obtained. The following features were found to characterize section staining of tannic acid mordanted sections. Tannic acid apparently blocks those sites that normally would be contrasted by uranyl acetate or some other staining compounds. Ribosomes remain unstained. Glycogen particles, on the other hand, were stained, whereas they are not in non-mordanted sections. In fact, glycogen was the only cytoplasmic component to be contrasted by the uranyl acetate, and collagen the only extracellular component. Several different section staining solutions gave the same staining patterns of examined cells and tissues. Specificity of the reaction thus seems to depend on the mordant rather than on the heavy atom section stain. Some other tested mordants, which have also been used in the light microscopical technique, did not give any useful new information.


Mechanisms contributing to ozone-induced bronchial hyperreactivity in guinea-pigs. The effect of ozone (3 ppm, 15-120 min) on bronchial reactivity in the guinea-pig was studied. Ozone induced marked (6-250-fold) bronchial hyperreactivity (BHR) to a range of inhaled, but not intravenous bronchoconstrictors. The degree of BHR was related to the duration of prior ozone exposure. The glutathione redox status was shifted to a more oxidized state in lung after 120 min ozone treatment, although no changes were found in the energy status of lung tissue, as judged by the concentrations of adenosine phosphates. Ascorbic acid pretreatment prevented BHR induced by 30 min ozone exposure. Neutral endopeptidase inhibitors elicited BHR to both substance P and histamine, but did not further enhance bronchoconstriction to substance P after ozone exposure for 120 min. Neither mepyramine, fentanyl, indomethacin nor a 5-lipoxygenase inhibitor (BW B70C), given prior to ozone exposure prevented the induction of BHR to histamine. Atropine or bilateral vagotomy reduced BHR after a 120-min, but not 30-min exposure to ozone. We conclude that in the guinea-pig, ozone induces non-specific, route-dependent BHR by oxidative injury, reducing airway NEP activity and enhancing the cholinergic and peptidergic component to bronchoconstriction. Neither cyclooxygenase nor 5-lipoxygenase products appear to play a role in ozone-induced BHR in this animal model.


Respiratory failure without pulmonary edema following injection of a glutamate agonist into the ventral medullary raphe of the rat. Injection of ibotenic acid (IA), a glutamate agonist, into the ventral medullary raphe (VMR; especially the nucleus raphe magnus) of the rat produced respiratory failure and death following a predictable course of events. The response to the IA injection was characterized initially by increased respiratory frequency and was followed by pulmonary arterial hypertension, systemic arterial hypoxemia, acidosis, and hypothermia. Within 90 min apnea occurred as a terminal event in all animals. Gravimetric, bronchoalveolar lavage protein, and histological analyses revealed no evidence of pulmonary edema. Intracerebral (VMR) pretreatment with PPP, a sigma receptor agonist, or scopolamine, a muscarinic cholinergic antagonist, prevented pulmonary failure and death even though postmortem histological analysis showed VMR cell loss and gliosis consequent to the cytotoxic IA injection. Based on the results of the study, it is suggested that the VMR has a role in regulation of pulmonary blood flow. Preliminary pharmacological studies suggested that a disruption of glutamatergic and cholinergic mechanisms mediates the lethal pulmonary phenomenon.


Paraoxon block of chloride conductance in cell R2 of Aplysia californica. In the presence of paraoxon, the amplitudes of chloride currents activated by acetylcholine (ACh) or gamma-aminobutyric acid (GABA) were reduced in cell R2 of Aplysia californica. IC50 values were 12 and 9.7 microM for ACh and GABA responses, respectively. Paraoxon did not affect resting membrane potential, input resistance, or chloride reversal potential. Both the slopes and maxima of ACh and GABA concentration-response curves were reduced by paraoxon, suggesting that paraoxon antagonism of these responses is not competitive. The antagonism of ACh and GABA responses by paraoxon was not related to inhibition of acetylcholinesterase.


Induction of expression of genes encoding transcription factors in the rat brain elicited by behavioral training. c-fos and zif/268 are regulatory genes encoding transcription factors able to influence gene expression directly. It has been shown repeatedly that expression of transcription factors correlates with different forms of cell activation, probably being functionally involved in the coupling of extracellular signals with long-term cellular responses. This study describes that c-fos and zif/268 mRNA accumulation, as measured by northern blot analysis, occurs in the rat hippocampus as well as the visual cortex following behavioral training of two-way active avoidance response.


Extendable words in nucleotide sequences. Previous statistical analyses revealed several peculiarities of nucleotide sequences that preclude their description by existing models and thus allow one to distinguish DNA and RNA sequences from random A,T,G,C-texts. This is a consequence of the unusual distribution of certain words in nucleotide sequences: while the distribution of (most) words is consistent with Markov models of small orders, the distribution of certain words cannot be described by any previous model (anomalies in distribution of homonucleotide/homopurine/homopyrimidine runs, complementary and mirror palindromes, and non-stationary words). In this work we introduce a probabilistic approach that is partly motivated by analogy with linguistics. We also describe another important feature of DNA/RNA sequences: anomalies in distribution of words of poor nucleotide composition. We show that some classes of these words are the major obstacle for the simple Markov description of nucleotide sequences.


Brief chemotherapy, involved field radiation therapy, and central nervous system prophylaxis for paranasal sinus lymphoma. Lymphoma of the paranasal sinus is a rare tumor characterized by bulky local disease, early systemic dissemination, and a propensity for central nervous system (CNS) spread. Treatment with radiation alone generally has been disappointing. Based on previous encouraging reports of initial brief chemotherapy followed by involved field radiation therapy (IFRT) for localized large cell lymphoma, four consecutive patients with paranasal sinus lymphoma were treated with 6 weeks of chemotherapy followed by IFRT and CNS prophylaxis. All patients had bulky localized disease and diffuse large cell lymphoma. Complete response was seen in all patients, and none have had a relapse (minimum follow-up, 25 months; range, 25 to 32 months). Chemotherapy and radiation therapy were well tolerated. One patient developed an osteogenic sarcoma in the radiation field 32 months after completion of therapy. Administration of early frequent chemotherapy followed by IFRT and CNS prophylaxis appears to be an effective treatment strategy for patients with localized large cell lymphoma of the paranasal sinuses.


Detection of a novel marker in the bronchial secretions of patients with non-small cell lung cancer using the 4B5 monoclonal antibody. A murine monoclonal antibody designated 4B5 was raised against the high molecular weight fraction of pooled sputum from patients with non-small cell lung cancer (NSCLC). Immunohistochemical staining indicated that 4B5 binds to histologically normal bronchial epithelium distant from tumor in 72% (39 of 54) of patients with NSCLC, but it binds to the primary cancer in only 13% (7 of 54) of the same patients. The antibody reacted less intensely with the bronchial epithelium in 16.6% (3 of 18) of autopsied patients without significant lung disease. The antigen recognized by 4B5 is a high molecular weight glycoprotein of more than 400 kilodaltons, judged by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis. Antigenic activity persisted after heating and resisted treatment with neuraminidase, but it was destroyed using protease and periodate. Multiple epitopes were present on each molecule recognized by 4B5. The determinants recognized by this antibody deserve additional study as possible markers of premalignant change in patients with NSCLC.


X-linked gene MIC5 codes for the L1 adhesion molecule recognized by monoclonal antibody R1. Monoclonal antibody (MoAb) R1 was generated by use of human-mouse hybrids containing only the long arm of chromosome X as the human component. It recognizes plasma membrane glycoproteins of 145 and 200 kd as well as a soluble protein of 195 kd. In this study, using a biochemical approach, we identified these glycoproteins as the human L1 adhesion molecule.


Heterozygote detection through bleomycin-induced G2 chromatid breakage in dyskeratosis congenita families. We determined the mean number of chromatid breaks per cell (b/c) in the bleomycin-treated lymphocytes of 10 patients with dyskeratosis congenita (DC) and 26 of their relatives to ascertain whether bleomycin sensitivity would distinguish DC heterozygotes from normal individuals. We observed a significantly higher mean number of chromatid b/c in DC patients and obligate heterozygotes (patients versus controls, p less than 0.0001; heterozygotes versus controls, p = 0.0076, Mann-Whitney rank-sum test). Unequivocal heterozygote detection was not possible owing to overlap of the b/c values of patients, heterozygotes, and controls, but our findings provided strong evidence of a link between autosomal recessive as well as X-linked recessive DC mutations and bleomycin sensitivity in homozygous, hemizygous, and heterozygous individuals.


Translocation (2;9)(p12;p23) in a case of acute leukemia with t(4;11)(q21;q23). Lack of rearrangement of the kappa and interferon gene loci. A case is reported of an adult male patient with acute leukemia characterized by the presence of the novel cytogenetic abnormality, t(2;9)(p12;p23), in addition to a t(4;11)(q21;q23). The immunophenotype of the blast cell population was consistent with immature early pre-B cell acute lymphoblastic leukemia (ALL) (TdT+,HLA-DR+,CD19+,CD24 +/-,CD10-) expressing myelo-monocytic antigens (CDw65,CD15). The genotype showed a clonal rearrangement of the immunoglobulin heavy chain locus. Because the immunoglobulin kappa (kappa) light chain gene is located on chromosome 2 at band p12 and interferon alpha (alpha) and beta (beta) map to chromosome 9p21-p22, rearrangements of these loci as a result of the t(2;9) were studied. There was no evidence for rearrangement of the region covering about 40 kilobases around the kappa locus when hybridized to C(kappa), the 3' kappa enhancer or the kappa deleting element. Only germline size restriction fragments were also found for the interferon alpha and beta genes. The patient's clinical features were typical for ALL associated with the t(4;11), including a high white blood cell count at presentation, hepatosplenomegaly, and a poor outcome. The potential significance of 2p and 9p abnormalities in addition to t(4;11) is discussed.


Modulation of trenimon-induced cytotoxicity by DT-diaphorase in isolated rat hepatocytes under aerobic versus hypoxic conditions. Trenimon belongs to a class of aziridinylbenzoquinone anticancer drugs that cross the blood-brain barrier. In this study we have investigated the molecular mechanisms for trenimon-induced toxicity in aerobic versus hypoxic conditions with the use of freshly isolated rat hepatocytes. The following evidence suggests the mechanisms for trenimon detoxification involves reduction by DT-diaphorase, while the cytotoxic mechanism involves macromolecular alkylation under hypoxic conditions as well as oxidative stress under aerobic conditions. (a) Hepatocyte cytotoxicity induced by trenimon (250 microM) under aerobic conditions ensued following an initial induction of cyanide-resistant respiration and partial oxidation of glutathione to oxidized glutathione. Trenimon reduction to the hydroquinone by the hepatocytes was rapid. Inhibition of hepatocyte DT-diaphorase by dicumarol increased trenimon-induced cytotoxicity by approximately 10-fold, and markedly inhibited hydroquinone formation. Furthermore, both cyanide-resistant respiration and oxidized glutathione formation were markedly increased, resulting in depletion of oxygen in the media. Trenimon reduction to the hydroquinone then occurred. This suggests that DT-diaphorase in normal hepatocytes prevents the formation of the semiquinone that causes cytotoxic protein alkylation and oxidative stress. (b) Hepatocyte cytotoxicity induced by trenimon (350 microM) under hypoxic conditions ensued following glutathione depletion without oxidized glutathione formation. Inactivation of hepatocyte DT-diaphorase by dicumarol under hypoxic conditions increased trenimon-induced cytotoxicity by approximately 3.5-fold and increased semiquinone radical levels 2-fold without affecting its reduction rate. This suggests that the cytotoxic mechanism involves protein alkylation by semiquinone radicals formed by reductases catalyzing a one-electron reduction of trenimon.


Endogenous receptor-bound urokinase mediates tissue invasion of the human lung carcinoma cell lines A549 and Calu-1. Macrophage colony-stimulating factor (CSF-1) increases the tissue invasive potential of the CSF-1 receptor-bearing lung carcinoma cell lines A549 and Calu-1 by increasing the number of endogenously bound urokinase-type plasminogen activators (u-PA)s on these cells. CSF-1, at concentrations which optimize invasion of A549 and Calu-1 cells into human amnion membranes (250 ng/ml), maximally augments the number of u-PA receptors occupied by endogenously produced urokinase. Preincubation of A549 and Calu-1 cells with the anti-u-PA monoclonal antibody MPW5UK (25 micrograms/ml) or with a 20- to 40-fold stoichiometric excess of fluid phase type 2 plasminogen activator inhibitor abrogates invasiveness, indicating that functionally active cell surface u-PA is essential for tissue invasion. In contrast, fluid phase type 1 plasminogen activator inhibitor (PAI-1, 5-15 units/ml) does not inhibit invasiveness unless preincubated with the amnion membranes. Inhibition of invasion by PAI-1 is abolished by presaturating the amnion membranes with antiitronectin monoclonal antibody (10 micrograms/ml) which prevents binding of PAI-1 to tissue-associated vitronectin. Binding of PAI-1 to tissue vitronectin is therefore a prerequisite for its inhibitory action. Thus, endogenously receptor-bound u-PA is the primary protease mediating CSF-1-induced tissue invasiveness of the lung carcinoma cell lines A549 and Calu-1.


Efficacy of an anti-CD7-ricin A chain immunoconjugate in a novel murine model of human T-cell leukemia. In vivo efficacy testing of monoclonal antibody-based drugs specific for human leukemias is hampered by the paucity of suitable animal models, due in part to the inability of many anti-human monoclonal antibodies to cross-react with antigens expressed in animal tissues or cells. Moreover, human leukemic cells have proven difficult to establish in immunosuppressed mice except as solid tumors. We report here the establishment of a murine model for human leukemia displaying features of human disease, such as growth of malignant cells and localization of such cells to lymphoid compartments, and the effective depletion of leukemic cells from these mice by an immunoconjugate. Human T-leukemia cells (CEM) injected into cyclophosphamide-pretreated NIH-III mice engrafted in all mice (n = 41), with CEM cells detected in the bone marrow, spleen, and blood 4 weeks after injection. There was no evidence of solid tumors. Treatment of CEM-engrafted mice with 4A2-RTA30, an immunoconjugate of an anti-CD7 monoclonal antibody and ricin A chain (RTA30), resulted in a 100- to 200-fold overall depletion of CEM cells from the spleen and the bone marrow (P less than 0.02). This depletion was specific and toxin-dependent, as a control immunoconjugate had no demonstrable effect (P greater than 0.5). Depletion of CEM cells was also observed after treatment with unconjugated anti-CD7 mAb, but this effect was not significantly different from controls (P greater than 0.1). Therefore, significant depletion of CEM cells required the presence of the ricin A chain moiety. Further investigations revealed that CEM cells recovered from NIH-III mice expressed less CD7 antigen, but remained sensitive to subsequent in vitro exposure to 4A2-RTA30. In conclusion, we have established a model for studying the efficacy of immunoconjugates and have successfully depleted human T-leukemic cells from lymphoid tissues in immunodeficient mice by treatment with an anti-CD7-RTA30 immunoconjugate.


Development of an in vitro model to study carcinogen-induced neoplastic progression of initiated mouse epidermal cells. Initiation and promotion in mouse skin carcinogenesis produce multiple benign tumors, squamous papillomas, but only a few squamous cell carcinomas. The spontaneous conversion from the benign to the malignant phenotype occurs over many months and in stages, but induced malignant conversion can be accomplished more rapidly by exposure of papilloma-bearing mice to mutagens or by transfection of papilloma cell lines with specific oncogenes. The analysis of genetic targets responsible for carcinogen-induced neoplastic progression would be facilitated by the development of in vitro models where the process is rapid, focal, and quantitative. To this end, primary newborn mouse keratinocytes were initiated in vitro by the introduction of the v-rasHa oncogene via a defective retrovirus. Recipient cells produce squamous papillomas and have a high proliferation rate in culture medium with 0.05 mM Ca2+, but fail to grow in medium with 0.5 mM Ca2+ which is permissive for growth of malignant keratinocytes. When v-rasHa-keratinocytes were exposed to mutagens in vitro, proliferative foci emerged after culture in 0.5 mM Ca2+ for 4 weeks. These foci stained intensely red with rhodamine stain, could be easily quantitated, and readily incorporated bromodeoxyuridine. Dose-response studies with several mutagens indicated that the number of foci increased with concentration to the point where excessive cytotoxicity developed. Mutagens varied in potency for producing foci in the following order: cis-diamminedichloroplatinum greater than or equal to benzo(a)pyrene diolexpoxide I greater than N-methyl-N'-nitro-N-nitrosoguanidine greater than or equal to 4-nitroquinoline-N-oxide greater than N-acetoxy-acetyl- aminofluorene. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate was inactive in the assay. A subset of cell lines derived from foci produced malignant tumors in vivo, while others were not tumorigenic. Analysis of DNA from cell lines and tumors revealed that most tumorigenic cell lines maintained the v-rasHa genome, whereas the viral sequences were deleted in nontumorigenic cell lines. Immunohistochemical analysis indicated that proliferative foci and quiescent v-rasHa keratinocytes expressed keratin 8, a marker of v-rasHa expression in cultured keratinocytes. Cells in foci, but not v-rasHa control cells, expressed keratin 13, a marker which is strongly associated with the malignant progression of skin tumors in vivo. This in vitro assay provides a quantitative model to study chemically induced focal neoplastic progression at the cellular level and to identify agents which may be selective for enhancing malignant conversion.


Potential autocrine role of insulin-like growth factor II during suramin-induced differentiation of HT29-D4 human colonic adenocarcinoma cell line. Suramin, a drug that binds to several types of growth factors, has been previously shown to induce the enterocyte-like differentiation of HT29-D4 human colonic adenocarcinoma cells, suggesting that growth factors are involved in such a process. Undifferentiated HT29-D4 cells release insulin-like growth factor II (IGF-II) into the culture medium that is totally complexed to heterogeneous IGF binding proteins (IGFBP) expressing high affinities for this growth factor (Kda = 0.02 nM and Kdb = 1.4 nM). These complexes do not allow IGF-II to bind to HT29-D4 cell surface type I IGF receptors, as evidenced by using 125I-IGF-II-IGFBP complexes. However, the addition of 40-100 micrograms/ml suramin, i.e., concentrations identical to the ones that are able to induce HT29-D4 cell differentiation, induces the release of IGF-II from IGF-II-IGFBP complexes, thereby allowing IGF-II to bind to the cell surface receptors. At such concentrations, suramin is indeed unable to alter IGF-II binding to HT29-D4 cells, a capacity that is observed only for concentrations higher than 200 micrograms/ml. Thus, suramin might have the unusual capacity to allow the establishment of an IGF-II autocrine loop involved in HT29-D4 cell differentiation. Consistent with this hypothesis is the fact that exogenously applied IGF-I (2.5 micrograms/ml) or agonist monoclonal antibody alpha IR-3 (2.5 micrograms/ml), which can bypass IGFBP present in the culture medium, induces part of HT29-D4 cell differentiation that is characterized by an important carcinoembryonic antigen release and the induction of numerous intercellular cysts with microvilli.


Cinnarizine-induced parkinsonism in primates. We describe the production of an experimental model of parkinsonism induced by cinnarizine (CNZ) in three healthy sylvanna monkeys. The drug produced a severe but reversible parkinsonism in all animals. After discontinuation of CNZ, all animals recovered but the oldest one was akinetic for 6 weeks. CNZ produced a persistent reduction in HVA and 5-HIAA levels in the CSF. Our data suggest a predominant presynaptic effect on DA and 5-HT neurons; and could account for the longstanding parkinsonism induced by calcium antagonist in some patients as well as the depression observed in these subjects.


Plasma beta-endorphin levels and natural-killer cells in two cases of congenital indifference to pain. We have investigated plasma beta-endorphin (beta-E), ACTH, and cortisol in two cases of congenital indifference to pain (CIP), a rare syndrome characterized by unresponsiveness to painful stimuli. As the two patients had frequent skin infections, we also studied lymphocyte response to mitogens in the absence or presence of beta-E. In addition, we explored a series of lymphocyte membrane antigens related to different aspects of the immune response, such as CD3+, CD4+, CD8+, B, NK Leu 7, Leu 9, and Leu 19, anti-interleukin-2 receptor (anti-TAC). Plasma beta-E levels in the two patients were significantly higher than in controls, whereas plasma ACTH and cortisol levels were normal. Lymphocyte response to the mitogen phytohemagglutinin was normal. The expression of Leu 7, Leu 9, and Leu 19, three antigens related to natural killer cells, was decreased by about 50%. The results indicate that in the two cases of CIP studied, high plasma beta-E levels are associated with a reduction in the expression of natural killer cells. This suggests that the two phenomena are specifically related to each other. These data represent further evidence of the possible pathophysiological relevance of the neuroendocrine-immune feedback.


Tracing the roots of ion channels.

Tracing the roots of ion channels. Two sets of recent findings draw our attention to questions concerning the origin of ion channels. First, there is sequence similarity among five classes of channels: voltage-gated channels, a putative Ca(2+)-activated K+ channel, cyclic nucleotide-gated cation channels, a putative Ca2+ channel for phosphoinositide-mediated Ca2+ entry, and a plant K+ channel/transporter. Like voltage-gated K+ channels, the most recently identified members of the superfamily share the basic design of one set of six potential membrane-spanning segments plus the H5 sequence; as such, they may resemble more closely the ancestral channel, which is likely to predate the separation of the animal and plant kingdoms. Second, several members of the ABC superfamily function as ion channels, even though they were previously known as transporters or enzymes. Did some ancestral enzymes subsequently acquire channel/transporter function? Or could it be the other way around? Aside from evolutionary considerations, enzymes and ion channels can no longer be treated as separate and nonoverlapping groups of proteins. When one molecule exhibits both functions, there are interesting mechanistic questions: How might the enzyme activity such as ATP hydrolysis be coupled to activation/regulation of the intrinsic channel activity? How might interactions between the permeant ions and the channel pore in turn regulate the enzymatic function of the same molecule? It seems possible that the latter is an extension of the observed coupling between permeant ions and the gating machinery of an ion channel (Swenson and Armstrong, 1981). Finally, the potential cross-regulation between channel activity and enzyme activity within the same molecule offers many intriguing possibilities for the integration of different cellular functions.


[Effect of 764-3 on bleomycin activation of alveolar macrophages]. The effect of pretreatment with 764-3, a huo xue hua yu medicine, on bleomycin A6 activation of alveolar macrophages of rats was observed. Results showed that bleomycin A6 could activate alveolar macrophages directly and that 764-3 could inhibit the activating effect of bleomycin A6. When alveolar macrophages are activated, they release not only PMN chemotactic factor, but also many destructive lysosomal enzymes, superoxide anions, etc, possibly resulting in serious injury to the lungs.


[Effect of 764-3 on histamine content in the lungs during inflammation and on the proliferation of lung fibroblasts]. The changes of histamine content in inflamed lungs induced by bleomycin, lung fibroblast proliferation enhanced by histamine and the effect of 764-3 on these parameters were investigated. Results showed that: 1) after intratracheal instillation of bleomycin for 7 and 21 days, the lung histamine content increased significantly, and 764-3 inhibited this increase of histamine content; 2) histamine enhanced the proliferation of lung fibroblasts in a dose- and time-dependent manner, and 764-3 inhibited the proliferation of lung fibroblasts in both normal and histamine-enhanced conditions. Results suggested that both inhibitory actions (direct inhibition of fibroblast proliferation and indirect action stemming from a reduction in the histamine content increase) might be involved in the antifibrotic mechanism of 764-3.


Interstrain cross-reactive idiotypes on monoclonal antibodies to an encephalitogenic myelin basic protein peptide. In order to assess the role of idiotype (Id) and the anti-Id network in murine experimental autoimmune encephalomyelitis (EAE), Id-bearing monoclonal antibodies (mAb) to human myelin basic protein (MBP) peptide acetyl 1-9, as well as mAb anti-Id, were developed in EAE-susceptible PL/J mice (H-2u). These mice recognize MBP residues acetyl 1-9 as an encephalitogenic determinant. Reactivities of PL/J Id-bearing mAbs to MBP and to MBP peptides were identical to those of mAbs generated against the same MBP peptide in EAE-resistant BALB/c mice (H-2d), even though isotypes of the mAbs differed. By using an inhibitory ELISA and immunoblotting, it was demonstrated that one PL/J mAb anti-Id recognized a public or framework Id, whereas another PL/J mAb-anti Id was directed to a private Id more restricted to the paratopic site. Two Id-bearing PL/J mAbs shared a cross-reactive Id (IdX) on the light chain, and an interstrain IdX was present on both the heavy and light chains of mAbs raised in PL/J and BALB/c mice to the same MBP peptide. The PL/J mAb anti-Id was capable of cross-regulating the production of Id-bearing mAbs by hybridomas across murine strains. These findings suggest that a restrictive family of germ-line genes encode for these Id-bearing antibodies to MBP peptide, irrespective of whether the MBP peptide is encephalitogenic in the murine strain immunized. Manipulation of the Id network may provide a means for modifying autoimmune demyelinating diseases of the central nervous system.


Hair follicle differentiation: expression, structure and evolutionary conservation of the hair type II keratin intermediate filament gene family. During hair follicle development several cell streams are programmed to differentiate from the cell population of the follicle bulb. In the hair cells, a number of keratin gene families are transcriptionally activated. We describe the characterization of the type II keratin intermediate filament (IF) gene family which is expressed early in follicle differentiation. In sheep wool, four type II IF proteins are expressed. One gene has been completely sequenced and the expression of three of the genes examined in detail. The sequenced gene encodes a 55 x 10(3) Mr protein of the type II keratin IF protein family, designated KII-9 in the new nomenclature we have adopted and described in the Introduction. The gene has a similar exon/intron structure to the epidermal type II keratin IF genes. In situ hybridization experiments show that the genes are expressed in the hair cortical cells but not in the cells of the outer root sheath, inner root sheath or medulla. During hair keratinocyte differentiation the type II IF genes are sequentially activated and coexpressed in the same cells. Expression is first detected in cells in the middle of the follicle bulb located near the dermal papilla and, subsequently, two of the genes are transcriptionally activated in the differentiating keratinocytes as they migrate upwards, in the upper part of the bulb. A fourth type II IF gene is activated later. The genes with the same expression pattern are also closely related in sequence and a number of conserved elements are present in the promoters of those genes, including a novel element which is also found in the promoter of a coexpressed type I IF gene and three other hair keratin genes.


Multimodality imaging and intracranial EEG display for stereotactic surgery planning in epilepsy. We describe a computer program for stereotactic surgery planning based on multimodality imaging and the display of intracranial EEG data in relation to anatomical data. The program is primarily designed to be used by surgeons to assist them in localizing brain structures and plan the safe and accurate insertion of surgical tools and depth electrodes. The mathematics underlying these concepts are briefly described. Estimates of the accuracy of the procedures are calculated and made available to the user. An extension of the program was developed to integrate intracranial EEG data with conventional images in order to help the neurologist and neurosurgeon in visualizing the EEG data in relation to the anatomy. It can be used to visualize the measured EEG voltages, or processed signals such as the application of the tetrahedral field calculation, which provides an extension of conventional bipolar EEG to 3 dimensions.


Sequential EEG mapping may differentiate "epileptic" from "non-epileptic" rolandic spikes. Sequential topographic mapping was performed to differentiate "epileptic" from "non-epileptic" rolandic spikes. Twenty-four children without any indication of organic brain lesion were divided into a group with epilepsy and a group without epilepsy. The group with epilepsy was subdivided into "classical BECT" (benign focal epilepsy of childhood with centro-temporal spikes) and "non-classical BECT." Sequential mapping of the rolandic spikes revealed two different topographic patterns: a pattern of stationary potential fields and a pattern of non-stationary potential fields. The topographic pattern of stationary potential fields was morphologically represented by a single spike-and-wave complex whereas that of non-stationary potential fields was morphologically represented by a "double" spike-and-wave complex. Among the non-stationary topographic patterns represented by a "double" spike, one specific sequence of changes of potential fields was found. This sequence started with a dipolar field, with the negative pole in the frontal region and the positive pole in the centro-temporal region, morphologically represented by the small first spike of the "double" spike-and-wave complex. This dipolar field, changes to a unipolar or dipolar field, with a negative potential field in the centro-temporal region and, sometimes, a simultaneous positive potential field in the frontal region, morphologically represented by the prominent rolandic spike. This characteristic pattern was found to be significantly related to classical BECT.


Regional coherence and the transfer of ictal activity during seizure onset in the medial temporal lobe. Epileptiform activity requires that large aggregates of neurons act synchronously. The process of neuronal synchronization during seizure onset was studied in the human medial temporal lobe by measuring the coherence of EEG activity. Records were obtained from 10 consecutive patients with hippocampal depth electrodes being evaluated for possible resective surgery. Coherence and phase spectra were calculated from all possible pairs of contacts in the medial temporal lobe of seizure onset using the method of Gotman applied to successive 6.4 sec epochs. Signals derived from adjacent contacts within definable brain regions were coherent during both the preictal and ictal period. Transitions in the level of coherence were measured between contacts presumed to span the boundaries of these regions. Time delays were measured early in the development of the seizure discharge but were not sustained. These time delays spanned the borders of regions of differing coherence, especially in the posterior hippocampus, and were interpreted to represent a transient increase in the functional linkage between structural elements. We conclude that the process of neuronal entrainment during seizure onset involves a transient interaction between brain regions but the maintenance of this interaction is not required for sustained seizure activity.


Automatic EEG interpretation: a new computer-assisted system for the automatic integrative interpretation of awake background EEG. A new computer-assisted system for automatic interpretation of the awake electroencephalogram (EEG) was developed. First, all the items necessary for EEG interpretation were determined in accordance with the procedure that a qualified electroencephalographer (EEGer) goes through for the visual inspection of the background EEG activity, and then each item was defined quantitatively. For the automatic interpretation, specific EEG parameters were determined for each item so that they could fit the graded judgement of the item by the qualified EEGer as closely as possible. These specific EEG parameters were actually calculated from periodograms obtained from the time series of EEG records of 14 patients with various neurological diseases. The automatic EEG interpretation system thus established was applied to the EEG data of these 14 subjects and to 3 additional EEGs, and the results were compared with those obtained through the visual interpretation by the EEGer. This automatic EEG interpretation was found to be in good agreement with the visual interpretation by the EEGer in most EEG records. In contrast with the previous automatic analyses of EEG which were focussed on certain aspects of EEG such as the dominant rhythm, the present system is unique in its capability of providing an integrative interpretation of the spontaneous awake EEG by taking into account all its features except for paroxysmal abnormalities.


Influence of lateral gaze on electroencephalographic spectral power. The effects of maintaining lateral gaze (as opposed to looking straight ahead) on electroencephalographic spectral power were tested in 12 right handed male subjects during eye opening (EO) and eye closure (EC). Our working hypothesis, based on Kinsbourne's paradigm, was that maintaining right lateral gaze activates the left hemisphere while maintaining left lateral gaze activates the right hemisphere, this activation resulting in a reduction in the spectral power over the hemisphere in question. Results showed that the variations in spectral power involved mainly the alpha frequency band. In the EC condition, the results were consistent with our working hypothesis: right lateral gaze produced a marked reduction in left hemispheric spectral power. In the EO condition, alpha power was constantly higher in the right hemisphere whether lateral gaze was maintained to the right or to the left. This can possibly be due to an attentional effect. Results are discussed with regard of the type of alpha rhythm and of the activation of cortical oculomotor centres. They shed light on the controversy concerning the existence of specific EEG correlates of cognitive activity, which preferentially involve each of the cerebral hemispheres.


Electroencephalographic studies in workers exposed to solvents or pesticides. The qEEG was studied in groups of young and old workers exposed to solvents (house and industrial painters) and in a group of workers exposed to pesticides. Three methods were used: a quantified visual scoring system, the neurometrics method and a multivariate analysis of mean frequencies. Using the visual assessment as well as the neurometrics method, the older painters showed more abnormalities than the younger painters and the pesticide exposed workers. The "profile" of abnormalities differed in all 3 groups. Changes of the mean spectral frequencies were mainly found in the subjects exposed to pesticides. In this study we concluded that the neurometrics method can be useful in neurotoxicological studies. With this technique minor changes in electrocortical function can be detected in seemingly normal workers. However, frequency parameters should be added to the power measures which are usually studied.


Effect of the cholinesterase inhibiting substance galanthamine on human EEG and visual evoked potentials. The action of galanthamine (GAL), a cholinesterase inhibiting substance, on resting EEG and on flash visual evoked potentials (VEPs) was tested in 9 healthy subjects. Alpha power was increased significantly in 4 of 8 subjects after the infusion of 10 mg, which provided a median inhibition of 47% of acetylcholinesterase in erythrocytes. Mean alpha frequency and peak alpha frequency decreased significantly in 5 of the 8 subjects by 0.22-0.98 Hz. Alpha power increase and alpha frequency decrease were not accompanied by changes in theta power. The amplitudes of the late components of the flash VEP were increased in 8 of 9 subjects receiving doses of 10-35 mg of GAL, while the early components remained unaffected. Increase of late VEP components was significantly correlated with the strength of cholinesterase inhibition. The synchronizing effect of GAL in these healthy volunteers obviously contrasts with the known desynchronizing effect of physostigmine in animal experiments.


Low contrast stimuli enhance PERG sensitivity to the visual dysfunction in Parkinson's disease. The pattern electroretinogram (PERG) was recorded at different contrast levels (96%, 71%, 47%) in 10 Parkinson's disease patients before and during dopaminergic monotherapy. The data were compared to a control group of 8 normal subjects recorded with the same procedure. PERG P50 latency progressively increased as contrast was decreased both in normal subjects and patients; however, this trend was much more pronounced in PD patients without therapy; consequently in this group the difference between P50 latency obtained with 96% and 47% contrast was statistically significant (P = 0.01, analysis of variance corrected by post-hoc Tukey test). By contrast this was not seen in the control group. Statistical analysis (Bonferroni's t test) showed at the 47% contrast level a significant P50 latency increase (P less than 0.01) in PD patients without therapy if compared with the control group. Dopaminergic monotherapy induced a P50 latency recovery in PD patients. We conclude that low contrast stimuli enhance PERG sensitivity to the visual dysfunction of PD patients. Moreover, the effects observed after therapy confirm that abnormal contrast response functions in PD patients are linked to dopaminergic deficiency.


Varying expressions of alerting mechanisms in wakefulness and across sleep states. Alerting stimuli, such as intense tones, presented to cats in wakefulness (W) elicit the orienting response (OR) and/or the acoustic startle reflex (ASR) in conjunction with elicited ponto-geniculo-occipital waves (PGOE) from the lateral geniculate body (LGB) and elicited waves from the thalamic central lateral nucleus (CLE). Alerting stimuli presented during rapid eye movement sleep (REM) and non-rapid eye movement sleep (NREM) also elicit PGOE. We presented tones in W, REM and NREM to determine whether CLE could be obtained in sleep and to examine the patterns of responsiveness of PGOE and CLE across behavioral states. Also, we recorded ASR and OR and compared the response patterns of behavioral and central correlates of alerting. The subjects were 7 cats; all exhibited spontaneously occurring waves in LGB and CL. All cats exhibited PGOE and 5 cats exhibited CLE in W, REM and NREM. PGOE and CLE showed less evidence of habituation than did ASR and OR. The pattern of responsiveness of CLE across behavioral states was different from that found for PGOE, and spontaneous CL waves were much rarer than the LGB waves. ASR was elicited in 5 cats during W trials, and in 3 cats during REM trials. OR habituated rapidly in W and did not occur in REM and NREM. The data indicate that central mechanisms of alerting function in sleep states as well as in W and suggest that CLE and PGOE reflect activity in mechanisms underlying cortical desynchronization and visual processes which may act in concert during alerting.


Frontal DC potentials in auditory selective attention. Selective dichotic listening during periods of 35 sec was associated with negative shifts of the cortical DC potential. Amplitudes of negative DC potentials had maxima in frontal, in particular, in anterior frontal records. The temporal pattern of negative DC potentials was different between the fronto-lateral records of the two hemispheres: in records from the right side, DC potentials declined during the 35 sec observation period, whereas they remained sustained in those of the left side. Different instructions ("attend left ear," "attend right," "attend both") and different levels of pitch separation between deviants and standards had no effects on frontal negative DC potential shifts, which are discussed in terms of higher order control of selective dichotic listening.


Relation of a negative ERP component to response inhibition in a Go/No-go task. Previous studies have suggested that a negative component (N2) of the event-related potential (ERP), whose peak latency is 200-300 msec after stimulus onset, may vary in amplitude depending on the neuronal activity required for response inhibition. To confirm this, ERPs were recorded in a Go/No-go paradigm in which subjects of one group (HI, n = 10) were asked to respond to Go stimuli with key pressing within a shorter period (less than 300 msec) than those of the other group (LI, n = 10) whose upper limit of the reaction time was relatively longer (less than 500 msec). All subjects had to withhold the Go response to the No-go stimuli without making overt muscle activities. The N2 component was recorded superposed on the initial descending limb of the P300 and other slow deflections, which were attenuated with a digital filter to measure the amplitude of N2. The N2 amplitude was significantly larger to the No-go stimulus than to the Go stimulus in both groups, but the N2 to the No-go stimulus was significantly larger in the HI group than in the LI group. These differences in N2 amplitude between conditions or groups were thought to be independent of other ERP components such as P300 and CNV. These results suggest that at least to some extent N2, which increased in amplitude when a greater effort was required to withhold the Go response, reflects the activity of a response inhibition system of the brain.


Comparison of different staining methods for polyvinylidene difluoride membranes. Several new staining methods for polyvinylidene difluoride membranes, including mercurochrome, silver and dimethylaminoazobenzene isothiocyanate staining were compared with Coomassie Brilliant Blue and gold staining. Of these, Coomassie was most versatile and completely compatible with ensuing microsequencing, immunostaining or other visualization methods, while gold and silver staining were more sensitive. Mercurochrome allows selective detection of sulfhydryl-containing proteins while dimethylaminoazobenzene isothiocyanate staining may allow quantitation of sequenceable protein.


Clinical, cytogenetic and immunological aspects in 4 cases resembling ataxia telangiectasia. Four cases resembling ataxia telangiectasia, all characterized by the absence of telangiectasias, are presented. Two are sisters while the other 2 are sporadic cases. The 2 sisters, aged 14 and 12 years, present a progressive neurological disease similar to that characterizing the Louis-Bar syndrome. The clinical picture in 1 of the sporadic cases, a girl aged 13 years, differs from the typical ataxia telangiectasia in having bilateral pyramidal signs in the lower limbs. The last case, a girl aged 8 years, presents an atypical clinical pattern characterized by a severe mental retardation, quite modest cerebellar signs and absence of involuntary movements. The results of the immunological and cytogenetic investigations are presented and discussed.


Partially reversible parkinsonism in Whipple's disease with antibiotherapy. Progressive parkinsonism developed in a 68-year-old man who had Whipple's disease. Extrapyramidal disturbances improved dramatically after introduction of antibiotherapy (trimethoprim-sulfamethoxazole).


Schistosoma mansoni: peritoneal plasmacytogenesis and polypoid transformation of mesenteric milky spots in infected mice. We have studied inflammatory reactions in the mesenteric tissue of mice infected with Schistosoma mansoni. Perivascular tissue contained diffuse infiltrates of macrophages, eosinophilic granulocytes and lymphocytes. Angiogenesis in the perivascular adipose tissue was associated with superficial plasmacytogenic foci. Polypoid structures were occasionally formed adjacent to inflammatory foci in the adipose tissue, organized around loops of capillaries, with terminal formation of a glomerular capillary network embedded in connective tissue, covered by plasmacytes. We conclude that these structures are specialized milky spots dedicated to active plasmacytogenesis and antibody secretion into the peritoneal cavity of schistosome-infected mice.


A protective monoclonal antibody with dual specificity for Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins. An IgM monoclonal antibody (Mab 36) which reacts with the circumsporozoite (CS) proteins of both P. falciparum and P. berghei was isolated from Plasmodium falciparum sporozoite-immunized mice. In assays of biological activity, Mab 36 induces the CS precipitation reaction with live sporozoites and blocks the invasion of hepatoma cells by sporozoites in vitro at concentrations much lower than those observed for previously reported CS protein-specific monoclonal antibodies. Mab 36 also provided complete protection against P. berghei sporozoite challenge in mice at low doses. Linear epitope mapping revealed that the epitope specificities recognized by Mab 36 are completely encompassed by other monoclonals previously shown to be associated in vivo with protection against P. falciparum or P. berghei sporozoite infection. These results suggest that the ability to make high-affinity IgM antibody to specific CS protein repeat epitopes may be important for eliciting protection against malarial infection.


Aprotinin: the ideal anti-coagulant?

Aprotinin: the ideal anti-coagulant? The serine proteinase inhibitor, aprotinin, significantly reduces transfusion requirements during open heart surgery. Whether this benefit is associated with an increased tendency to thrombosis has not been studied. We investigated the effect of aprotinin in an experimental arterial thrombosis model. In 17 male Sprague-Dawley rats, the infrarenal aorta was replaced with 1.0-mm diameter PTFE grafts of varying lengths. The time to graft occlusion, recorded by palpation, Doppler ultrasound and a distal bleeding test, was 20.2 +/- 1.8 min, 35.8 +/- 6.1 min and 43.7 +/- 6.6 min for grafts of 10, 7.5 and 5.0 mm respectively (r = -0.98, p less than 0.05). Following PTFE graft placement 24 Sprague-Dawley rats were given saline (n = 6), aprotinin (n = 6), heparin (n = 6), and heparin + aprotinin (n = 6). The time to occlusion was significantly prolonged in the aprotinin group (71.7 +/- 20.4 min vs. 20.2 +/- 1.8 min, p less than 0.05). The time to thrombosis for heparin + aprotinin and heparin alone was also significantly prolonged (p less than 0.05). Prothrombin times (PT) were 21.9 +/- 3.0 s for control, 29.4 +/- 6.2 s for aprotinin, 40.7 +/- 2.5 s for heparin and 39.9 +/- 14.5 s for heparin + aprotinin (p less than 0.05 vs. control for all values). Bleeding time was not prolonged with aprotinin (3.0 +/- 0.9 min vs. 2.9 +/- 0.7 min). The bleeding time was 18.9 +/- 4.1 min for heparin + aprotinin and 22.5 +/- 2.3 min for heparin alone (p less than 0.05 vs. control for both values).(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of the RNA processing enzyme RNase III from wild type and overexpressing Escherichia coli cells in processing natural RNA substrates. 1. A precursor to small stable RNA, 10Sa RNA, accumulates in large amounts in a temperature sensitive RNase E mutant at non-permissive temperatures, and somewhat in an rnc (RNase III-) mutant, but not in an RNase P- mutant (rnp) or wild type E. coli cells. 2. Since p10Sa RNA was not processed by purified RNase E and III in customary assay conditions, we purified p10Sa RNA processing activity about 700-fold from wild type E. coli cells. 3. Processing of p10Sa RNA by this enzyme shows an absolute requirement for a divalent cation with a strong preference for Mn2+ over Mg2+. Other divalent cations could not replace Mn2+. 4. Monovalent cations (NH+4, Na+, K+) at a concentration of 20 mM stimulated the processing of p10Sa RNA and a temperature of 37 degrees C and pH range of 6.8-8.2 were found to be optimal. 5. The enzyme retained half of its p10Sa RNA processing activity after 30 min incubation at 50 degrees C. 6. Further characterization of this activity indicated that it is RNase III. 7. To further confirm that the p10Sa RNA processing activity is RNase III, we overexpressed the RNase III gene in an E. coli cells that lacks RNase III activity (rnc mutant) and RNase III was purified using one affinity column, agarose.poly(I).poly(C). 8. This RNase III preparation processed p10Sa RNA in a similar way as observed using the p10Sa RNA processing activity purified from wild type E. coli cells, confirming that the first step of p10Sa RNA processing is carried out by RNase III.


Methotrexate local injection for unruptured tubal pregnancy: an alternative to laparotomy? Fifty-nine women with early unruptured tubal pregnancy were treated by a single local injection of methotrexate at laparoscopy. All 59 patients underwent the procedure without any adverse reaction, 47 (80%) of them needing no laparotomy. Twelve patients required a laparotomy for reasons such as rising beta-hCG levels and abdominal pain with or without rising levels of beta-hCG. Only one patient ruptured the tube. None of the women needed a blood transfusion. We found tubal patency in 19 out of 21 patients at follow up hysterosalpingography. Eleven pregnancies were subsequently reported, one of them tubal. The appearance of the injected tube was absolutely normal in three patients, one at cesarean section and two at repeated laparoscopy. No peritubal adhesions were observed. We suggest that this new technique is a safe and effective alternative to laparotomy in a patient with an early unruptured tubal pregnancy.


Development of cycles for lyophilization. In order to find the freeze-drying cycle for the effective drying of a given biological entity or a material of biologic origin by sublimation of ice in vacuo, numerous time-consuming, trial and error preliminary studies have been required. Following a series of studies using different shelf temperatures, -30 degrees, -10 degrees, 0 degrees, +10 degrees and +20 degrees C, and elapsed times of 1,500, 2,000, 2,700, 4,000 and 5,500 minutes, plug characteristics and contents of residual moistures of freeze-dried 3 ml samples of 2% serum albumin were determined. Using the statistical method of least squares, geometric curves were fitted to the plot of times versus residual moistures for the several shelf temperatures. The equations for the fitted curves were used to construct a table showing the contents of residual moistures at 300 minute intervals. This table was used for developing successful drying cycles for several concentrations of serum albumin, dilute solutions of interferon, and serum containing HLA antibodies.


Minor neurological dysfunction from birth to 12 years. I: Increase during late school-age. To study the hypothesis that the frequency of minor neurological dysfunction (MND) stabilizes around the age of nine years, two groups of the Groningen Perinatal Project (GPP) were re-examined at 12 years. The study group (N = 174) was selected on the basis of the presence of MND at nine years; the control group comprised 172 neurologically normal children. The hypothesis was rejected: extrapolation of the findings to the total GPP population showed that the over-all rate of MND increased. Control children who developed MND were mainly boys who had been neurologically abnormal at birth or were born preterm and/or had experienced an adversity in combination with asphyxia. Interval complications between nine and 12 years were related to the emergence of MND.


Minor neurological dysfunction from birth to 12 years. II: Puberty is related to decreased dysfunction. To determine whether puberty is related to decreased minor neurological dysfunction (MND), 174 children from the Groningen Perinatal Project who had had MND at nine years were re-examined at 12 years. No signs of MND could be demonstrated in 39 of the children, 33 of whom showed at least three signs of puberty. The presence of minor physical anomalies was associated with persisting MND. The authors hypothesise that puberty is related to a decrease in MND, and discuss the role of hormonal changes in relation to the decrease in minor signs. Re-examination at 14 years will be necessary to confirm this hypothesis, since 68 per cent of the children had not yet reached puberty. Children with MND reached puberty no later than those without.


Phosphorylation of ligand-gated ion channels: a possible mode of synaptic plasticity. Most neurotransmitter receptors examined to date have been shown either to be regulated by protein phosphorylation or to contain consensus sequences for phosphorylation by protein kinases. Neurotransmitter receptors that mediate rapid synaptic transmission in the nervous system are the ligand-gated ion channels and include the nicotinic acetylcholine receptors of muscle and nerve and the excitatory and inhibitory amino acid receptors: the glutamate, GABAA, and glycine receptors. These receptors are multimeric proteins composed of homologous subunits which each span the membrane several times and contain a large intracellular loop that is a mosaic of consensus sites for protein phosphorylation. Recent evidence has suggested that extracellular signals released from the presynaptic neuron, such as neurotransmitters and neuropeptides as well as an extracellular matrix protein, regulate the phosphorylation of ligand-gated ion channels. The functional effects of phosphorylation are varied and include the regulation of receptor desensitization rate, subunit assembly, and receptor aggregation at the synapse. These results suggest that phosphorylation of neurotransmitter receptors represents a major mechanism in the regulation of their function and may play an important role in synaptic plasticity.


Connections: heart disease, cellular electrophysiology, and ion channels. Our purpose in this article is to examine the hypothesis that both myocardial disease and ischemia can alter the electrophysiologic function of the ion channels responsible for the cellular electrical activity of the heart. Changes in the intracellular and extracellular milieus occur during ischemia and can alter the electrophysiology of several species of ionic channels and the cellular electrophysiologic activity of cardiac myocytes. Included are 1) changes in extracellular [K+] and pH and in intracellular [Na+], [Ca2+], and pH; 2) accumulation of noxious metabolic products such as lysophosphatidylcholine; and 3) depletion of intracellular ATP. Finally, ischemia or disease (e.g., hypertrophy) can alter the electrophysiology of at least two types of K+ channels, the A-like channels underlying the transient outward current and the inward rectifier, by mechanisms that apparently do not involve alteration of either the intra- or extracellular milieus. Findings suggest that the expression of cardiac A-like channel function can be altered by hypertrophy and that at least one intrinsic conductance property of the inward rectifier can be altered by ischemia. We speculate that the control of expression, function, and regulation of cardiac ion channels can be affected at the molecular level by heart disease and myocardial ischemia.


Secretagogue-evoked time-course changes on pancreatic juice secretion in the anaesthetized rat. 1. In the present time-course study, we have examined the interactions between the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and the synthetic gut hormones, cholecystokinin-octapeptide (CCK-8) and secretin on pancreatic juice secretion in anaesthetized rat. 2. Administration of either TPA (10(-8) mol kg-1 hr-1), secretin (100 pmol kg-1 hr-1) or CCK-8 (150 pmol kg-1 hr-1) in the anaesthetized rat resulted in marked time-course increases in pancreatic juice flow, amylase secretion and total protein output compared to saline controls. The effect of secretin on juice flow was more pronounced and sustained compared to the smaller responses obtained with either CCK-8 or TPA. Similarly, CCK-8 evoked increases in protein output and amylase secretion compared to the responses obtained with either secretin or TPA. 3. Simultaneous infusion of TPA with either CCK-8 or secretin resulted in a marked reduction in pancreatic juice flow, total protein output and amylase secretion compared to the responses obtained with either CCK-8 or secretin alone. 4. Administration of polymyxin B (10(-8) mol kg-1 hr-1), a protein kinase C inhibitor with either TPA and CCK-8 or TPA and secretin caused a partial reduction of the inhibitory effect of TPA on CCK-8 and secretin-evoked secretory responses. 5. The present study further implicates the involvement of protein kinase C in the modulation of CCK-8 and secretin-induced pancreatic juice secretion in the anaesthetized rat.


Effective portal insulin delivery with enzyme-protected capsules in pancreatectomized pigs. 1. Plasma concentrations of insulin, C-peptide, glucagon and glucose were measured in surgically pancreatectomized pigs given insulin into the colon directly and in enteric peptidase-resistant (methacrylic acid copolymer-encapsulated) form. 2. Following introduction of insulin-containing capsules, plasma insulin concentration rose from 2.7 +/- 0.1 microU/ml to 110.9 +/- 51.9 microU/ml in the portal vein, and from 2.6 +/- 0.1 microU/ml to 26.9 +/- 7.3 microU/ml in the systemic circulation. Corresponding portal and systemic values after direct (non-encapsulated) insulin instillation were 28.2 +/- 15.9 microU/ml to 44.8 +/- 13.0 microU/ml and 7.5 +/- 2.6 microU/ml to 15.2 +/- 2.5 microU/ml respectively. Insulin concentrations peaked at 75 min in the group as a whole and between 60-90 min in individual animals. Absorption was most pronounced in pigs given aprotinin (a trypsin inhibitor) with insulin. 3. Plasma portal vein glucose concentrations fell from 76.2 +/- 8.9 mg/dl to 31.1 +/- 3.2 mg/dl 150 min after encapsulated insulin administration. Corresponding systemic glucose levels were 84.5 +/- 11.0 mg/dl and 37.0 +/- 1.4 mg/dl. 4. Colonic administration of insulin in methacrylic acid coated capsules results in peak portal and systemic insulin levels 60-90 min after administration. Co-administration of aprotinin enhances the fraction of insulin absorbed.