Adhesion receptor profile of thymic B-cell lymphoma. Primary thymic B-cell lymphoma is clinically characterized by aleukemic, highly aggressive local growth, infrequent distant metastasis, and infrequent secondary lymph node involvement. VLA-1 to VLA-6 are cell surface molecules binding to matrix molecules such as collagen, fibronectin, epiligrin, and laminin. VLA-4 additionally binds to VCAM-1 and ICAM-2, thus mediating intercellular adhesion. Other molecules involved in cell/cell adhesion are LFA-1 (CD11a/CD18), Mac-1(CD11b/CD18) and their ligand ICAM-1 (CD54), p150,95 (CD11c/CD18), LFA-3 (CD58), CD44, and LECAM-1. Twenty-three tumors, together with normal lymphoid tissue, were immunohistochemically examined to investigate the expression pattern of these molecules in thymic B-cell lymphomas and in their putative normal counterparts, namely thymic medullary B cells. Thymic B-cell lymphomas consistently lacked VLA-1,-2,-3,-5,-6, and CD11b, expressed ICAM-1 in 21 of 23 cases but were heterogenous for VLA-4, LFA-1, CD11c, LFA-3, CD44, and LECAM-1. Presence of LFA-1 correlated with LFA-3 expression (P = 0.029). The receptor profile of thymic B-cell lymphoma was reminiscent of the expressional status of normal thymic medullary B cells in some aspects but deviated in others: Assuming that, in terms of differentiation, thymic B-cell lymphoma is related to the asteroid variant of thymic medullary B cells, a propensity to down-regulate/lose VLA-4, CD11a, CD44, and LECAM-1 would have to be supposed in conjunction with a tendency to overexpress ICAM-1 and LFA-3. Sclerosis as an inconsistent phenomenon in thymic B-cell lymphoma was absent in 8 of 23 tumors. Presence of sclerosis correlated with LECAM-1 expression of the tumor cells (P = 0.038). Recent studies suggest that a locally growing/aleukemic phenotype of a B-cell neoplasia might be determined by the phenotype VLAs-, LFA-1+, ICAM-1+, CD44-, and LECAM-1-. Our data corroborate this view.


Morphologic changes in basal cells during repair of tracheal epithelium. Basal cells are differentiated with respect to junctional adhesion mechanisms and play a role in attachment of columnar epithelium to the basal lamina. Although much is known about nonciliated and ciliated cell differentiation during the repair process after injury, little is known about the basal cell. We studied the morphology of basal cells and quantitated junctional adhesion structures during repair of tracheal epithelium exposed to toxic cotton smoke. Ten adult ewes were given a smoke injury to a portion of the upper cervical trachea and were killed at 4, 6, 8, 10, and 18 days after injury for morphometric studies. At 4 days, there was a stratified reparative epithelium over the basal lamina, which was two to four cells in depth. The basal cells were identified by their hemidesmosome (HD) attachment to the basal lamina. Basal cells were about 69% larger than controls and flattened rather than columnar. The amount of HD attachment was 192% greater than controls. In contrast, volume density of cytokeratin filaments had decreased about 47%. Basal cells had returned to normal numbers and size and a columnar shape by day 18. The amount of desmosome (D) and HD attachment and volume density of cytokeratins had also reached control levels by day 18. These data indicate that morphology of basal cells changes during the initial stages of reparative regeneration but returns to normal by 18 days. Morphologic changes appear to reflect changes in size of the cell associated with cell division rather than differentiation of recently divided basal cells.


Continuous spectrophotometric assay of protein tyrosine phosphatase using phosphotyrosine. A continuous activity assay for protein tyrosine phosphatases (PTPs), employing phosphotyrosine (P-Tyr) as a substrate, has been developed and applied to measure the activities of two purified enzymes, namely, the full length T-cell protein tyrosine phosphatase (TC PTP) and its truncated form (TC delta C11 PTP). The reaction was followed by changes in ultraviolet absorption and fluorescence resulting from the dephosphorylation of P-Tyr. Both enzymes obey Michaelis-Menten kinetics, with Km = 304 microM, Vmax = 62,000 units/mg for TC PTP and Km = 194 microM, Vmax = 73,000 units/mg for TC delta C11 PTP. The D- and L-forms of P-Tyr are equally effective as substrates. The optimum pH for both enzymes is 4.75. The known effectors of PTPs have the predicted effects on catalytic activity.


Detection and quantitation of recombinant granulocyte colony-stimulating factor charge isoforms: comparative analysis by cationic-exchange chromatography, isoelectric focusing gel electrophoresis, and peptide mapping. Routine quantitation of recombinant human granulocyte colony-stimulating factor charge isoforms in the purified protein product requires development of a reliable analytical method. In this report, isoelectric focusing gel electrophoresis, peptide mapping, and cation-exchange high-performance liquid chromatography are compared and evaluated in the analysis of charge isomers that may be present in the recombinant factor. Due to a lack of sensitivity and reliability, isoelectric focusing gel electrophoresis and peptide mapping are not recommended. However, peptide mapping can distinguish aberrant peptides with differences in charges and provide separation for subsequent structural characterization. By this approach, an N-terminally blocked formylmethionyl species was identified to be the minor charge isoform in the purified preparations of recombinant human granulocyte colony-stimulating factor. In contrast to electrophoresis and peptide mapping, a strong cationic-exchange chromatographic procedure was found to be the most selective, sensitive, and reproducible analytical method. The sensitivity and reliability of the method were evaluated and validated using the formylmethionyl isoform and several deamidated analogs (Gln----Glu) made by site-directed mutagenesis. Recombinant human granulocyte colony-stimulating factor preparations contain a very low to undetectable level of the formylmethionine isoform and have no detectable deamidated isoforms.


Substance P, thyrotropin-releasing hormone, and monoamine metabolites in cerebrospinal fluid in sleep apnea patients. The cerebrospinal fluid (CSF) concentrations of thyrotropin-releasing hormone (TRH), substance P (SP), 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), and 3-methoxy-4-hydroxyphenyl glycol (MHPG) were measured in 15 consecutive patients with the sleep apnea syndrome (SAS) and in healthy control subjects. Second measurements were performed 6 months after surgical treatment in 10 of the patients. The mean (+/- SD) concentration of TRH-like immunoreactive material (TRH-LIM) (pg/ml) did not differ significantly between patients with SAS (8.1 +/- 2.8) and control subjects (7.5 +/- 2.2). However, postoperatively, this concentration was increased in the six clinically cured patients with SAS, from 6.9 +/- 2.7 to 9.4 +/- 1.6 (p less than 0.03). Substance P-like immunoreactive material (SP-LIM) was higher in untreated patients with SAS than in control subjects: 19.2 +/- 6.7 versus 14.4 +/- 4.2 fmol/ml (p less than 0.02), and the level remained high after operation in the group treated surgically. The HVA, 5-HIAA, and MHPG concentrations were similar in patients with SAS and control subjects, and no consistent changes were found postoperatively. The CSF deviations in TRH-LIM and SP-LIM concentrations in the patients may reflect a primary central nervous system defect or they may be secondary to intermittent nocturnal hypoxia, progressive hypercapnia, and/or sleep fragmentation. In this sense, both these systems may be markers of SAS-SP as a "trait" marker and TRH as an indicator of the current state.


[Stem cell factor/c-kit interaction in primordial germ cell, melanoblast and hematopoietic progenitors]. Mutation at S1 or W loci are characterized by lacks of pigmentation, gametogenesis and hematopoiesis. Stem cell factor and its receptor, which is encoded by c-kit proto-oncogene, play an important role in the survival and proliferation of these primitive cells. Primordial germ cell is maintained and expanded on cells transfected with membrane-bound SCF gene. Pigmentation of mouse embryo is influenced by administration of monoclonal antibody for c-kit product, ACK 2, because of inhibition of melanoblast migration to epidermal tissue. Moreover, hematopoietic progenitors are considered to be maintained and expanded in liquid culture in the presence of SCF and other growth factors. All of these primitive cells express c-kit product and the direct action of SCF is expected. However, two types of SCF, soluble form and membrane-bound form, exist and the physiological significance of these forms in vivo remain unsolved.


[Treatment of elderly patients with hematological malignancies]. Treatment of elderly patients with hematological malignancies is difficult and a matter of controversy. Low responsiveness to therapy and high risk of mortality have been reported. The risk of chemotherapeutic death increases after age 60, and an age-adjusted chemotherapy schedule is needed. In stage III and IV Hodgkin's disease, for example, an age-adjusted COPP regimen may be adopted. Many non-Hodgkin lymphomas (NHL) of elderly patients have a slow course. However, for intermediate to high grade aggressive NHL, dose-reduced CHOP regimen, or non- or low-dose methotrexate-containing programs like BECALM, CNOP, and low dose-ACOP-B are acceptable. MACOP-B regimen with G-CSF may be used for patients under age 65. For the treatment of elderly patients with AML, it is reported that a reduced-dose DAT regimen is better than the standard dose for inducing CR in patients older than 60. In elderly AML patients over 60, the dose-adjustment reported by Mori, or low-dose cytarabine with G-CSF, is recommended. Information about elderly patients with acute lymphoblastic leukemia is scarce. Aggressive treatments like L-17 M regimen are not tolerable by elderly patients, and a combination chemotherapy consisting of vincristine and prednisolone is recommended.


[Quality of life for older people under oncological treatment]. Recently, an argument over the medical treatment of older patients has arisen in the field of clinical oncology. In the past oncological treatment has been geared to young and middle-aged people. Currently, oncological treatment takes the peculiarities of older people into consideration. Firstly, it highlights the multidimensionality of Quality of Life (QOL) as an important element, with subjectivity the second element. This notion has gained a worldwide consensus. The domain of "multidimensionality" consists of: 1. Physical status and functional abilities 2. Psychological status and well-being 3. Social interaction 4. Economic status and factors With these factors in mind, different questions are asked, depending on the condition of each patient, in order to let the patient form his/her own judgment on possible medical treatment. However, it is sometimes difficult for older people to make such judgments due to problems such as depression or their diminishing mental ability. Also, it is impossible to define life satisfaction indiscriminately without regard to individual outlook. QOL for older people is generally characterized by "individuality" which includes: 1) A person's integrity; 2) independence; and 3) autonomy. Though these elements are taken into account in adopting the above-mentioned methodology, the field of clinical oncology's two concepts of QOL still apply in principle. Flexibility to put emphasis on different elements of the domain will be necessary. Even though QOL for older people is a matter of subjective judgment, the "sound judgment of professionals" (objective and scientific judgment of caregivers) can be adopted in cases when there is difficulty in communication.(ABSTRACT TRUNCATED AT 250 WORDS)


[High-dose adjuvant chemotherapy with peripheral blood stem cell transplantation for breast cancer with poor prognosis--a pilot study]. Three patients with breast cancer with poor prognosis were treated with high-dose chemotherapy (HD-CT) and peripheral blood stem cell transplantation (PBSCT) as adjuvant treatment. After radical mastectomy, the consolidation chemotherapy with Adriamycin 50 mg/m2, Cyclophosphamide 1,000 mg/m2, Vincristine 1.0 mg/m2 and Methotrexate 200 mg/m2 with Leucovorin rescue was started. Recombinant human granulocyte colony stimulating factor (rhG-CSF) was also added for early recovery from myelosuppression. This combination chemotherapy was given every 3 weeks for 3 courses, and after the 2nd and 3rd courses, peripheral blood stem cells (PBSC) were collected and cryopreserved. HD-CT with Cyclophosphamide 2,000 mg/m2/day, Thio-TEPA 200 mg/m2/day, and Etoposide 300 mg/m2/day, were administered for 3 consecutive days, and after 48 hours of last doses, frozen-thawed PBSC (6.4-8.9 x 10(4)/kg of CFU-GM) were administered. rhG-CSF was also added. HD-CT and PBSCT were well tolerated, recovery from myelosuppression of the HD-CT was very quick and no serious side effects were observed.


[Anticancer effect and side effect of arterial chemoembolization using cis-diamine-dichloroplatinum (II)/4-0-tetrahydropyranyl-adriamycin-lipiodol (CTL) suspension on hepatocellular carcinoma]. Seventeen patients with hepatocellular carcinoma were treated by intraarterial injection of CTL suspension. The doses of CTL suspension, CDDP and THP(mean +/- SD)/injection were 4.1 +/- 1.6 ml, 81.9 +/- 31.6 mg and 13.5 +/- 5.2 mg, respectively. The therapy was given once in 10 patients, twice in 6 and 4 times in one. Over 50 per cent reduction in tumor size was obtained in 5 patients (30%). Fifty or more % decrease in serum alpha-feto-protein (AFP) levels was observed in 3 of 7 patients (43%) with the initial serum AFP level of more than 200 ng/ml, Fever, abdominal pain, nausea and vomiting were noted in most cases. However, they disappeared within 2 weeks after therapy was completed. No severe complications were encountered except one case of a liver abscess which healed by administration of antibiotics. No severe changes in laboratory data were observed. This study suggests that a new method of intraarterial injection must be developed to enhance the therapeutic effect even more, in addition to an increased injection dose of CDDP/THP-LPD and higher concentration of CDDP and THP in LPD.


Transient hypothyroxinaemia associated with developmental delay in very preterm infants. In 563 surviving very preterm (less than 32 weeks gestational age) and/or very low birthweight (less than 1500 g) infants the relationship between neonatal thyroxine concentration and psychomotor development at 2 years of age (corrected for preterm birth) was studied. A significant association was found between low neonatal thyroxine concentration and a negative score on the three milestones of development. These findings do not support the view that transient hypothyroxinaemia in preterm infants is harmless.


Histamine release and morphological changes in basophilic granulocytes of atopic asthmatics induced by antigen, anti-IgE and Ca ionophore A 23187. Morphological changes of basophils from atopic asthmatics were compared among antigen, anti-IgE and Ca ionophore A23187 stimulation. 1. Antigen induced rapid and marked increase of histamine release from basophils compared with Ca ionophore A23187. 2. The decrease in number of basophils following stimulation with the agents was significantly higher in antigen stimulation than in Ca ionophore A23187 stimulation. 3. The increased ratio of short to long axis diameter (L/Sb ratio) of the cells, which shows an increased motility of basophils, was significantly higher in antigen stimulation. The ratio did not change by stimulation with Ca ionophore A23187. 4. Stimulation of basophils by Ca ionophore A23187 induced marked increase in mean diameter (MD) of the cells. Activation of basophils by anti-IgE was similar to that by antigen, but slower in start and shorter in duration compared with antigen. The results show that an increase in motility is essential for release mechanism of chemical mediators from basophils in antigen and anti-IgE stimulation, but not in Ca ionophore A23187 stimulation.


Anti-peptide antibodies reactive with epitopic domains of porcine amelogenins at the C-terminus. This was an immunological investigation of the processing of porcine amelogenins in situ. Rabbit and rat anti-peptide sera reacted specifically with the hydrophilic segment of the intact amelogenins at the C-terminus. The immunogens used were the synthetic peptides: (a) C13 composed of PATDKTKREEVDC and (b) C25 composed of MQSLLPDLPLEAWPATDKTKREEVD. These peptides correspond to the C-terminal 12- and 25-residue segments of porcine amelogenin, respectively. Cystine was introduced at the C-terminus of C12 for KLH-binding (C13). Western blot analysis disclosed that: (i) both rabbit and rat anti-C13 sera reacted selectively with the 25-kDa porcine amelogenin and three other minor components (27, 22 and 18 kDa); (ii) anti-C25 peptide sera, additionally, reacted with the 23-kDa amelogenins (a degradation derivative of the 25-kDa protein, lacking the 12-residue segment at the C-terminus) and as trace components, 20-, 16- and 14-kDa moieties. Importantly, all the proteins reactive with the anti-C13 serum were concentrated in the outer secretory enamel adjacent to the ameloblasts, decreasing significantly in the underlying inner secretory enamel. Immunohistochemical studies applying the anti-peptide sera to the developing tooth germs of a minipig also confirmed the localization of reactivity in the outer secretory region. Neither anti-peptide serum reacted with porcine non-amelogenins, serum proteins nor dentine matrix proteins at the dilutions tested. however, it was found that both the anti-C13 and C25 sera reacted with human keratin.(ABSTRACT TRUNCATED AT 250 WORDS)


Regulation of secretion by vasoactive intestinal peptide in isolated perfused rat submandibular glands. The isolated, perfused gland was used to examine the regulation of saliva volume and protein content by vasoactive intestinal peptide (VIP). In the absence of other secretagogues, VIP produced a modest, sustained saliva flow with a biphasic dose-response curve in which saliva volume was greatest at 1 nM VIP (28.5 +/- 3.8 microliters in the first 5 min, n = 4) but reduced at lower and higher concentrations. The protein concentration in saliva released in response to VIP (0.86 +/- 0.13 micrograms/microliters) was substantially higher than with 30 nM acetylcholine (0.06 +/- 0.02 micrograms/microliters) or 1 nM substance P (0.30 +/- 0.05 micrograms/microliters). During the first 5 min of stimulation, VIP and substance P were synergistic in terms of volume and protein content whereas inclusion of VIP did not increase acetylcholine-stimulated flow in the first 5 min but produced a higher sustained flow over the next hour. After stimulation with acetylcholine, subsequent addition of VIP transiently enhanced saliva volume and protein content in a monophasic, dose-dependent manner with effects at 1 pM VIP and higher. The responses were different for VIP compared with other cAMP-mobilizing agents and the involvement of multiple VIP receptor subtypes was suggested from experiments in which a VIP antagonist blocked the VIP enhancement of saliva volume but not the increase in protein.


Immunofluorescent lectin binding patterns and glycoprotein co-localization in the developing murine molar tooth. Fluorescein-conjugated lectins were used in conjunction with antibodies to laminin, tenascin and amelogenin to investigate saccharide expression in the developing tooth germ. At the bud stage, peanut agglutinin (PNA) binding demonstrated residues that may be D-galactose-(beta 1----3)DGalNAc, and this staining occurred after the expression of tenascin. Only the cap-stage enamel organ suprabasal cells and the enamel knot stained intensely with Ulex europeus agglutinin-I, but not Lotus tetragonolobus agglutinin, implying the transient presence of blood group H type I oligosaccharides. At the late stages of amelogenesis, enamel synthesis is preceded by en bloc loss of inner enamel basement membrane components. Before this, Bandeiraea (Griffonia) simplicifolia--I (BSL-I) staining was lost from postmitotic ameloblasts, suggesting that a glycosylated species is initially removed. Additionally, PNA was co-localized with amelogenin protein, suggesting that it may express beta-D-galactosyl sequences. These results indicate that the glycosylation patterns of matrix components during odontogenesis may be important as they vary in a manner similar to that of the well-known glycoproteins.


Primary nonkeratinized epithelial ('conjunctival') orbital cysts. The types of orbital cysts that are most familiar to ophthalmologists are epidermoid and dermoid cysts, both of which are lined by keratinized stratified squamous epithelium. We studied six patients who had orbital cysts lined by nonkeratinized epithelium that resembled normal conjunctiva. Compared with epidermoid and dermoid cysts, these primary nonkeratinized cysts tend to cause symptoms later in life, occur preferentially in the superonasal aspect of the orbit, and are less likely to be associated with underlying bony changes.


Visual symptoms associated with choroidal neovascularization. Photopsias and the Charles Bonnet syndrome. One hundred consecutive patients with macular choroidal neovascularization were studied in a cross-sectional fashion. Evidence of bilateral choroidal neovascularization was present in 31 patients. Among the 100 subjects, 59% related a history of seeing flickering or flashing lights (photopsias) in the affected eye or eyes. The colors varied, but in 59% of instances the lights were white. Twelve subjects experienced formed hallucinations (Charles Bonnet syndrome); in nine (75%) of these patients, the sequelae of choroidal neovascularization were bilateral. Symptoms that are commonly attributed to vitreoretinal tractional phenomena as well as neurologic and/or psychiatric disease are also frequently encountered in patients with macular degeneration associated with choroidal neovascularization.


Recovery of mitochondrial DNA from blood leukocytes using detergent lysis. Mitochondrial DNA (mtDNA) was isolated from leukocytes contained in whole blood of cattle. Leukocyte membranes except the nuclear envelope were solubilized in a buffer that contained 1% Triton X-100. After sedimentation of cell nuclei, mtDNA was purified from the cell lysate by organic solvent extraction and ethanol precipitation. Approximately 5 micrograms of mtDNA was recovered from 400 ml of whole blood, a quantity sufficient for routine DNA cloning procedures or for detailed restriction mapping studies. mtDNA isolated with this method is a suitable substrate for several DNA-modifying enzymes. Thus, preparation of mtDNA from blood by detergent lysis provides a noninvasive alternative to tissue biopsy for characterization of mitochondrial genotypes in studies of evolutionary genetics and population dynamics.


Nucleotide sequence and expression of a cDNA encoding rabbit liver cytosolic serine hydroxymethyltransferase. A rabbit liver cDNA library in phage lambda gt10 was screened using a portion of the coding sequences for rabbit cytosolic serine hydroxymethyltransferase (amino acids 244-420) that had been amplified by PCR, with total rabbit liver RNA as a template. A clone of 2.3 kb (pUS1203) was isolated and the nucleotide sequence showed that it contained an open reading frame of 1452 bp, which coded for serine hydroxymethyltransferase and was flanked by 155 bp at the 5' end and 653 bp at the 3' end. The full-length cDNA was cloned into an expression vector and transfected into COS-1 cells. Serine hydroxymethyltransferase activity was increased by 33% in the transfected cells and a new protein band of the appropriate size was seen by SDS/PAGE analysis of proteins extracted from transfected cells. The protein sequence for rabbit cytosolic serine hydroxymethyltransferase derived from the cDNA nucleotide sequence was compared with three other derived or known prokaryotic and eukaryotic sequences. An overall sequence similarity of 34% was noted between all four sequences, whereas the similarity between the rabbit cytosolic and mitochondrial isoforms was 62%.


Age-dependent decrease in the amount of eukaryotic initiation factor 2 in various rat tissues. Recent studies have suggested that the decline in protein synthesis that occurs in rat liver and brain during development and aging is associated with a decrease in the activity of eukaryotic initiation factor 2 (eIF-2). One way in which eIF-2 activity could be decreased in tissue extracts would be through a decrease in the activity of the GDP exchange factor, eIF-2B. In the present study, the activity of eIF-2B was measured in tissue extracts and was found to be less in older than in younger rats. Thus a decrease in eIF-2B activity could account for part of the decrease in protein synthesis that occurs during aging. Another way in which eIF-2 activity could be decreased would be through a decrease in amount of the protein. Therefore the amount of eIF-2 in various tissues was quantified by protein immunoblot analysis. We found that the amount of eIF-2 relative to total protein tended to fall with increasing age. Furthermore, eIF-2 content was directly proportional to the rate of protein synthesis in the tissues examined. Finally, slot-blot analysis of polyadenylated RNA revealed no significant change in the relative abundance of eIF-2 alpha mRNA with age. The last-mentioned experiments suggest that the synthesis of eIF-2 may be regulated through changes in the deficiency of translation of eIF-2 alpha mRNA rather than through changes in gene transcription.


Structure and expression of the Drosophila ubiquitin-52-amino-acid fusion-protein gene. Ubiquitin belongs to a multigene family. In Drosophila two members of this family have been previously described. We report here the organization and expression of a third member, the DUb52 gene, isolated by screening a Drosophila melanogaster genomic library. This gene encodes an ubiquitin monomer fused to a 52-amino acid extension protein. There are no introns interrupting the coding sequence. Recently, it has been described that this extension encodes a ribosomal protein in Saccharomyces, Dictyostelium, and Arabidopsis. The present results show that the 5' regulatory region of DUb52 shares common features with the ribosomal protein genes of Drosophila, Xenopus and mouse, including GC- and pyrimidine-rich regions. Moreover, sequences similar to the consensus Ribo-box in Neurospora crassa have been identified. Furthermore, a sequence has been found that is similar to the binding site for the TFIIIA distal element factor from Xenopus laevis. The DUb52 gene is transcribed to a 0.9 kb mRNA that is expressed constitutively throughout development and is particularly abundant in ovaries. In addition, the DUb52 gene has been found to be preferentially transcribed in exponentially growing Drosophila cells.


Lipoprotein (a) promotes plasmin inhibition by alpha 2-antiplasmin. Plasmin inhibition by alpha 2-antiplasmin (alpha 2AP) is regulated by the vascular components fibrin(ogen) fragments, plasminogen and lipoprotein (a). Kinetic analysis demonstrates that CNBr-derived fibrinogen fragments completely protect plasmin from alpha 2AP. Plasminogen and 6-aminohexanoic acid decrease the rate of inhibition by 5- and 10-fold respectively. These studies show that CNBr-derived fibrinogen fragments and 6-aminohexanoic acid bind plasmin kringle(s) with binding constants of 2 micrograms/ml and 120 microM respectively, and that plasminogen binds to alpha 2AP with an affinity of 0.5 nM. The unmodulated inhibition is not effected by the presence of lipoprotein (a), but in the presence of protective CNBr-derived fibrinogen fragments the rate of inhibition is increased by the presence of the lipoprotein. The kinetics demonstrate that lipoprotein (a) binds to CNBr-derived fibrinogen fragments with an affinity of 4 nM, displacing plasmin from the protective surface. In addition, tissue-type plasminogen activator and trypsin inhibition by alpha 2AP is not slowed by the presence of CNBr-derived fibrinogen fragments or plasminogen (Pg), respectively. These kinetics suggest that the initial reversible interaction between plasmin and alpha 2AP is mediated by binding of the inhibitor to the kringle 1 domain of plasmin, with a reversible inhibition constant (Ki) of 5.0 x 10(-10) M. Under conditions where this kringle-inhibitor interaction is blocked, the reversible inhibition still occurs between the plasmin and alpha 2AP, but the initial Ki is increased to 5.0 x 10(-9) M. These data suggest that, in the circulation, plasmin inhibition by alpha 2AP may be down-regulated by fibrin, fibrin(ogen) fragments and Pg, but up-regulated by lipoprotein (a) in the presence of fibrin or fibrin(ogen) fragments. The lipoprotein (a)-mediated promotion of plasmin inhibition may provide an additional mechanism by which the lipoprotein impairs fibrinolysis and promotes atherosclerosis.


CD11b is a calcium-dependent epitope in human neutrophils. Human neutrophils expressing complement receptor 3 (CR3) were treated with various concentrations (0.04-10 mM) of Ca2+/Mg(2+)-chelating agent EDTA and the expression of CD11b, the CR3 alpha chain antigenic epitope, was examined using monoclonal antibodies and flow cytometry. EDTA caused a dose-dependent decrease in the reactivity of two anti-CD11b monoclonal antibodies, Leu15 and IOM1. The reduced expression of CD11b in EDTA-treated cells was partly restored by the addition of Ca2+ ions whereas the addition of Mg2+ ions had no effect on CD11b level. The expression of the CR3 beta chain epitope, CD18, was markedly decreased only by 10 mM EDTA. These results suggest that the CD11b epitope may be associated with the Ca(2+)-binding domains of CR3 alpha chain and its recognition by antibodies depends on the presence of bound Ca2+.


Effects of Taxotere on murine and human tumor cell lines. Taxotere (RP 56976, NSC 628503), an analog of taxol, is an inhibitor of depolymerisation of microtubules and is currently in Phase I clinical trials. Comparisons of the cytotoxicities of Taxotere and taxol have been studied on several murine (P388, SVras) and human cell lines (Calc18, HCT116, T24, N417, KB). Taxotere was found more potent than taxol (1.3-12 fold), a result which could be explained by its higher affinity than taxol for microtubules. In agreement with its postulated mechanism of action, Taxotere is more cytotoxic on proliferating than on non proliferating N417 cells and does not inhibit cellular DNA, RNA and protein synthesis. Taxotere gives partial cross resistance on P-glycoprotein resistant P388/DOX cell line, in contrast to taxol which gives a complete cross resistance. On the other hand, no cross resistances were observed on Calc18/AM and P388/CPT5 cell lines, bearing modified activities of topoisomerase II and topoisomerase I, respectively. These results underline the higher cytotoxic activity of Taxotere compared to taxol, and the lack of cross resistance of that class of agent with the topoisomerase I and II-related multidrug resistance phenotypes.


Ozonation of lysozyme in the presence of oleate in reverse micelles of sodium di-2-ethylhexylsulfosuccinate. Ozone is shown to react with lysozyme in reverse micelles formed by 0.1 M sodium di-2-ethylhexylsulfosuccinate and 1.2-3 M water (pH 7.4) in isooctane solvent. The reaction of ozone is assessed by the oxidation of tryptophan residues in the protein to N-formylkynurenine. Cosolubilization of oleate in lysozyme-containing reverse micellar solutions at concentrations of 0.5-10 mM results in a progressive inhibition (19% to 82%) of the oxidation of tryptophan residues with a concentration for 50% inhibition around 2 mM. At this concentration of oleate, the magnitude of inhibition is independent of the micelle size and concentration, the overall interfacial area of reverse micelles, and the amount of ozone employed. These findings are discussed in terms of competitive reactions of ozone with unsaturated fatty acids and proteins in the lung lining fluid and in biological membranes.


The sulfhydryl reagent N-ethylmaleimide induces hyperphosphorylation on tyrosine residues in the Jurkat T-cell line. Tyrosine protein kinases have been shown to be functionally involved in regulation of cellular signalling, proliferation and transformation. The activity of tyrosine protein kinases is counterbalanced by phospho tyrosine phosphatases that maintain constitutively low levels of protein phosphotyrosine in most cells. In this study the effect of N-ethylmaleimide on the protein tyrosine phosphorylation was tested in Jurkat T-cells. Treatment of intact cells for 5-10 mins with 50-100 microM N-ethylmaleimide resulted in a dramatic increase in phosphorylation on tyrosine residues. Phosphoaminoacid analysis revealed an up to ten-fold increase in the content of phosphotyrosine. N-ethylmaleimide blocked the phospho tyrosine phosphatases activity of immunoprecipitated CD45 while in a kinase assay N-ethylmaleimide did not affect the 32P-gamma-ATP phosphorylation of substrates. The N-ethylmaleimide-induced hyperphosphorylation was reversed by treatment with 2 mM dithiotreitol. It is concluded that N-ethylmaleimide offers a novel useful tool for identification of substrates for tyrosine protein kinases and for studies on phosphotyrosine-dependent protein interactions.


Synthesis of hydrolytic enzymes during production of tylosin by Streptomyces fradiae. The exposure of a wild-type tylosin producing strain of Streptomyces fradiae to mutagenic agents resulted in the isolation of several tylosin over-producing strains. Examination of three mutants, T4310, 612 and 3204 showed that improved tylosin production was associated with increased hydrolytic enzyme activity and cell growth. The wild-type strain showed lower levels of hydrolytic activity including, protease, amylase, lipase and esterase activities and attained a lower cell density than the mutants.


In situ hybridization: a valuable tool in diagnostic pathology. In situ hybridization or hybridohistochemistry has evolved in recent years in a new histologic modality. In situ hybridization (ISH) can be used for the detection of DNA (DISH) or RNA (RISH). The potential diagnostic value within a pathologic setting are well recognized. In this review paper, we summarize the use of DISH in a pathologic setting for the detection of chromosomal aberrations and localization of DNA-viruses like cytomegalovirus and Epstein Barr virus. RISH which is still in a more experimental stage can be applied for the localization of RNA-virus, like human immunodeficiency virus. However, the most important application of RISH will be the detection of gene-expression at the level of mRNA. Potentially this has many applications especially in early diagnostics of neoplastic tissues. Finally, we have summarized some pitfalls which may hamper the introduction of in situ hybridization for diagnostic purposes and some future developments in ISH.


Participation of lipopolysaccharide-binding protein in lipopolysaccharide-dependent macrophage activation. Only recently has the mechanism for lipopolysaccharide (LPS) recognition by macrophages been elucidated. In contrast to many ligand receptor interactions, the interaction of LPS with its receptor, CD14, on myeloid cells is greatly enhanced by prior complexation of LPS with LPS-binding protein (LBP), a recently discovered plasma glycoprotein. LBP is found in normal serum or plasma in the 5 to 10 micrograms/ml range. In plasma, it reacts rapidly but transiently with LPS. LPS-LBP complexes then react with CD14 bearing cells. Blocking CD14 with monoclonal antibodies or removal of LBP from plasma blocks the ability of the cells to react with LPS-LBP complexes and also blocks release of cytokines and other mediators from the cells. In the normal lung, bronchoalveolar lavage fluid contains low levels of LBP. However, during acute lung injury, LBP levels may rise by transudation and enhance activation of alveolar macrophages to release injurious mediators. Description of this pathway for LPS recognition by macrophages and other leukocytes offers the possibility of developing new reagents to block LPS recognition and prevent the development of endotoxemia.


Altered adhesion molecule expression and endothelial cell activation accompany the recruitment of human granulocytes to the lung after segmental antigen challenge. Mounting evidence suggests that inflammatory cells recruited to the lung can contribute to the pathogenesis of asthma. The factors governing the activation and recruitment of circulating cells to the lung remain unknown, but an early step in this process is the interaction of adhesion molecules on circulating cells with those on endothelial cells. We used a segmental antigen challenge model followed 18 h later by bronchoalveolar lavage (BAL) to study granulocyte recruitment to the lung in 14 allergic subjects. Using immunofluorescence and flow cytometry, we determined the expression of the adhesion molecules CD11b, L-selectin (LECAM-1), and VLA-4 on BAL and peripheral blood granulocytes. Total cell count and percentages of recovered eosinophils and basophils were significantly increased in BAL fluids from antigen-challenged segments. Compared with their peripheral blood counterparts, CD11b expression was increased 2- to 3-fold on BAL eosinophils, basophils, and neutrophils (n = 9, P less than 0.05). In contrast, L-selectin expression was significantly decreased on BAL cells (n = 3 to 4, P less than 0.05). Similar phenotypic changes were observed on all three cell types, and on neutrophils recovered from saline-challenged control lung segments. In two subjects, VLA-4 alpha (CD49d) expression on BAL eosinophils was 78 +/- 5% of that seen on peripheral blood eosinophils. Because ELAM-1 (endothelial leukocyte adhesion molecule-1, E-selectin) expression occurs during allergic inflammation and is shed after endothelial activation, we used a sensitive enzyme-linked immunosorbent assay to analyze BAL supernatants for a soluble form of this molecule (sELAM-1).(ABSTRACT TRUNCATED AT 250 WORDS)


CSF monoamine metabolites in old age dementias. Cerebrospinal Fluid (CSF) levels of the main metabolites of monoamines (MHPG, 5-HIAA, and HVA) were measured in patients with early onset (AD) and late-onset (SDAT) Alzheimer's disease, vascular dementia (VD), and elderly controls. Psychobehavioral assessment was carried out by means of MMSE and GBS. Mean MHPG levels did not differ from controls; 5-HIAA was lower in VD when compared to both controls and SDAT. HVA was decreased in AD, SDAT, and VD with respect to controls. Significant correlations between HVA and psycho-behavioral parameters were observed in SDAT and VD groups, whereas no relationship was documented in AD. The SDAT group was divided in SDAT-A (age at onset: greater than 65 less than or equal to 80 yr) and SDAT-B (age at onset: greater than 80 yr). SDAT-A had significantly lower CSF HVA values than SDAT-B (165 +/- 64 vs 235.7 +/- 85). SDAT-B HVA levels were similar to those observed in controls. Correlation analysis between HVA and neuropsychological variables was significant in SDAT-A, but not in SDAT-B. These results might support the evidence of SDAT heterogeneity.


Carbohydrate changes in colon carcinoma. The colonic epithelium preferentially expresses only certain types of carbohydrate antigens. The most prevalent ones are the lacto-series type 1 (Gal beta 1,3GlcNAc) and type 2 (Gal beta 1,4GlcNAc) chain blood group-related substances, which can reside on both glycolipids and glycoproteins, and mucin-type O-linked structures such as Tn, T, and sialosyl-Tn, which reside exclusively on glycoproteins. This review focuses on the tissue expression in the colon of those carbohydrate tumour-associated antigens which have been most extensively studied and for which the epitope structure has been defined.


Cell surface carbohydrate in cell adhesion. Sperm cells and leukocytes bind to their target cells through specific oligosaccharide ligands. During the past few years it has become increasingly evident that specific oligosaccharides in glycoproteins/glycolipids may play a central role in mammalian cell adhesion. This review deals with two of the best studied systems, the interaction of sperm cells with the Zona pellucida layer surrounding mammalian eggs, and the binding of leukocytes to endothelial cells. In the sperm cell-Zona pellucida system, it is known that a specific oligosaccharide(s) in the Zona pellucida glycoprotein 3 is responsible for binding. In the leukocyte-endothelial cell interaction, sialyl Le(x), sialyl Le(a), sulfatides, and other less well-defined sugar structures are involved in cellular binding. Oligosaccharides involved in these adhesion events may turn out to have clinically important applications.


Glycosylation of neural cell adhesion molecules of the immunoglobulin superfamily. Cell adhesion molecules (CAMs) are believed to play key roles during morphogenesis. In this review we focus on neural CAMs belonging to the immunoglobulin superfamily. Data concerning distribution, expression pattern, structure and function of these CAMs are accumulating these years. In general, little is known about the importance of glycosylation for the function of these CAMs. The neural cell adhesion molecule, NCAM, is probably the best described CAM, and this molecule exhibits special carbohydrate characteristics, e.g. NCAM is polysialylated. Glycosylation of NCAM seems to be regulated during development and to influence the adhesive function of the molecule. Structure, function and glycosylation of NCAM are described in detail.


Selective inhibition of HIV replication by adriamycin in macrophages but not in lymphocytes. Adriamycin (ADR) is an anticancer drug commonly used in the treatment of HIV-related cancers. Due to its effect on DNA metabolism, ADR might be able to modulate HIV replication in monocyte-macrophages (M/M), resting cells potentially less sensitive to the toxic effect of this drug. Thus, we assessed the efficacy of ADR against HIV replication in both lymphocytes and M/M. We further investigated the mechanism(s) of action of ADR and its potential synergistic activity with zidovudine (AZT) or alpha-interferon (IFN alpha). ADR consistently inhibited viral replication in M/M: 50% viral inhibition was obtained with 0.005 micrograms/ml ADR, while greater 90% viral inhibition was obtained with 0.05 micrograms/ml ADR. No cell toxicity was seen in M/M at concentrations up to 0.5 micrograms/ml. No anti-HIV activity was shown by ADR in lymphocytes at concentrations up to 0.05 micrograms/ml, that is also the toxic dose 50% (TCID50 for these cells). ADR neither inactivates HIV virions nor affects HIV binding with CD4 receptors. No inhibition of HIV reverse transcriptase by ADR was found at concentrations at least 2,000-fold greater than the 50% HIV inhibitory concentration in M/M. Molecular analysis by polymerase chain reaction (PCR) suggests that ADR substantially affects virus DNA production at concentrations that inhibit viral replication. Finally, late stages of HIV replication were not affected by ADR. At least additive effects of the association ADR + AZT and ADR + IFN alpha were obtained against de novo HIV infection of M/M.(ABSTRACT TRUNCATED AT 250 WORDS)


Human immunodeficiency virus replication: modulation by cellular levels of cAMP. HIV infection is associated with qualitative and functional immune deficiencies. It has been shown that the in vitro infection of CD4+ cells with HIV was associated with sustained elevation of cAMP and cGMP. In the present report the role of cAMP on HIV replication in MT-4 cells was investigated. The MT-4 cells were infected with HIV (strain 3b), in the presence or absence of agents that increase intracellular levels of cAMP, through different mechanisms. At selected times postinfection, HIV replication was measured by reverse transcriptase activity or HIV P24Ag in culture supernatants. Forskolin (FK, an activator of adenylate cyclase 1-100 microM), Isobutyl-methylxanthine (IBMX, a phosphodiesterase inhibitor, which indirectly increases intracellular levels of cAMP, 30-100 microM) and dibutyryl (db) cAMP (0.1-10 microM) enhanced HIV replication, in a dose-dependent manner. FK, IBMX, and db cAMP enhanced HIV replication by 2- to 10-fold, 4- to 7-fold, and 2- to 6-fold, respectively. Intracellular levels of cAMP were measured by radioimmunoassay and were also enhanced. Since cAMP exerts its catalytic effects through activation of protein kinase (PK) A the effect of H-8 (a specific inhibitor of the cAMP dependent PK A) on HIV replication was simultaneously examined. The H8 at doses of 0.1 to 10 microns inhibited HIV replication by 25 to 99.9%. Moreover H9 inhibited HIV replication in peripheral blood mononuclear cells by more than 90%. The replication of HIV appears to be a cAMP-dependent event, and PK A could possibly be a target for the development of anti-HIV therapies.


[A patient with epilepsy presenting developmental regression after seizures--consideration of the mechanism from the findings of CT-scan and SPECT]. We report a 6-month-old female infant with frequent brief seizures who subsequently showed developmental regression. CT-scans right after her seizures revealed enlargement of bilateral subdural space more on the right side with an evidence to suggest destruction of the blood brain barrier in enhanced CT-scan, CT-scan, one month after the onset, showed diffuse brain atrophy. 123I-SPECT, which was performed 11 days after cessation of her seizures, still showed diffuse hypoperfusion predominantly in the right temporal area, and it returned to normal 3 months after the onset. We discussed the mechanisms of developmental regression in this case from the neuroradiological findings.


12-O-tetradecanoylphorbol 13-acetate induced differentiation in human lung squamous carcinoma cells. Three human lung squamous carcinoma cell lines (NX002, CX140 and CX143) demonstrate features of squamous differentiation including involucrin synthesis and competence to form cornified envelopes. 12-O-Tetradecanoylphorbol 13-acetate inhibits growth of these cell lines and this growth inhibition is associated with enhanced differentiation.


Control of H-2 expression in transformed nonhaemopoietic cells by autocrine interferon. The relationship between autocrine interferon (IFN) production and the expression of class I Major Histocompatibility Complex (MHC) membrane glycoproteins in vitro was investigated in a panel of murine transformed cells of nonhaemopoietic origin. The panel included 11 cell lines of H-2Kb haplotype derived from fibrosarcomas, carcinomas and melanoma, and from transformed fibroblasts. IFN activity was detected in the conditioned medium of nine cell lines; fibrosarcomas were among the high IFN producers, while the non-producers were a melanoma clone and a lung carcinoma cell line. A significant correlation was found between IFN production and the expression of H-2K/D glycoproteins, thus suggesting that long-term maintainment of MHC glycoprotein expression in vitro could be mediated by self produced IFN. Two IFN producer cell lines, MN/MCA1 and R80/17, were cultured in the presence of a blocking antiserum against IFN-alpha/beta: a significant decrease in H-2b expression was observed, thus indicating the existence of an autocrine IFN circuit. Taken together these findings suggest that release of IFN is a frequent event among transformed nonhaemopoietic cells, and that self-produced IFN contributes to the regulation of MHC antigen levels in solid tumours.


Antibodies for denatured human H-ferritin stain only reticuloendothelial cells within the bone marrow. Human H-ferritin homopolymer was denatured in sodium dodecyl sulphate and injected in mice to obtain antibodies for dissociated H-subunit. The antisera and Moabs obtained were specific for the denatured H-chain with no cross-reactivity with assembled ferritins in immunoblotting experiments. In contrast the Moabs for native recombinant H-ferritin are specific for the assembled ferritin molecules with weak cross-reactivity with the denatured H-subunits. The epitope recognized by one of the anti-denatured H-chain Moabs was mapped on the C-terminal helix of ferritin. The antibodies were used to study H-ferritin conformation in cells. In immunocytochemistry experiments the antibodies for denatured H-ferritin stained HeLa and K562 cells weakly, with a different intensity and pattern to those obtained with anti-native H-ferritin antibody. In human bone marrow smears the anti-denatured ferritin antibodies stained only reticuloendothelial cells, and did not recognize the H-ferritin rich immature erythroblasts. It is concluded that assembled and denatured H-ferritins are immunogenically distinct, and that erythroid and reticuloendothelial cells within the bone marrow contain H-ferritin in different conformations.


Surface marker expression in acute myeloid leukaemia at first relapse. Surface markers were studied at first relapse in 66 cases of acute myeloid leukaemia (AML), using a panel of five monoclonal antibodies directed to CD13, CD14, CD15, CD33 and CD34 antigens. At time of relapse, there was increased expression of CD33 (P = 0.002) and CD34 (P = 0.0001), and decreased expression of CD13 (P = 0.004) and CD15 (P = 0.0001) antigens by comparison to initial diagnosis. There was no strict correlation with the FAB classification. However, CD13 and CD33 expression changes preferentially affected granulocytic leukaemias. At relapse, CD14 and CD34 were significantly more expressed in monocytic than in granulocytic AML (P = 0.01 and 0.003 respectively). In a multivariate analysis, CD34 expression was associated with a low CR rate (P = 0.001) and short survival (P = 0.05), whereas CD15 expression was associated with long survival (P = 0.0004). These results suggest that AML tends to relapse with a less differentiated phenotype than observed at diagnosis and that AML with less differentiated phenotype is of poor prognosis after first relapse, as also observed at diagnosis.


Conserved natural humoral immunity to thyroglobulin in patients with multiple myeloma. We studied humoral immunity to human thyroglobulin (hTg) during the course of multiple myeloma (MM). In this report, we describe the anti-hTg antibody activity in the sera of patients with MM. Among 63 sera tested, 28 (44%) had IgG anti-hTg autoantibodies (aAb), 16 (25%) exhibited IgM aAb, and six (9%) had IgA anti-hTg aAb. For the majority of sera the anti-hTg autoantibody activity was associated with more than one immunoglobulin class. IgG anti-hTg antibodies were observed in 9/11 patients with IgA MM and in 19/40 patients with IgG MM. The IgM anti-hTG antibody activity was found in the sera of 11 patients with IgG MM. These results show that the anti-hTg activity in these patients is associated with residual polyclonal immunoglobulins. However, in the serum of one patient presenting a double monoclonal gammopathy (IgG and IgA lambda MM), the anti-hTg activity was carried by both the IgG lambda and the IgA lambda molecules, suggesting that in this case the activity was due to the monoclonal immunoglobulin itself. We also studied the epitopic specificity pattern of all these anti-hTg aAb. Only three sera recognized one antigenic region on hTg, suggesting that the majority of the anti-hTg aAb in MM patients were directed against antigenic regions other than those recognized by our panel of murine mAb. In conclusion, our results demonstrated that humoral immunity to hTg is maintained in MM patients. These data contrast with the well-documented suppression of immunity to foreign, especially bacterial, antigens described in MM.


Normal aggregations of glycoprotein IV (CD36)-deficient platelets from seven healthy Japanese donors. Since glycoprotein IV (GPIV) has been shown to play an important role in the interaction of platelets with collagen and thrombospondin, the aggregation and secretion of GPIV-deficient platelets were examined. Using a binding assay with monoclonal 125I-OKM5 antibody against CD36 antigen and crossed immunoelectrophoresis of the solubilized platelets against anti-GPIV antibody, the platelets from seven (4.1%) out of 170 healthy Japanese donors were found to be deficient in GPIV. The GPIV-deficient platelets showed normal aggregations in response to collagen as well as ADP, epinephrine, arachidonic acid and thrombin in comparison with GPIV-positive platelets. Polyclonal anti-GPIV antibody aggregated GPIV-positive platelets but not the GPIV-negative ones. The F(ab')2 fragments of the anti-GPIV antibody competitively inhibited the anti-GPIV-induced aggregation, but did not affect the collagen-induced aggregation of GPIV-positive platelets. These results suggest that the deficiency of GPIV does not affect platelet aggregability.


Effects of arotinolol on exercise capacity and humoral factors during exercise in normal subjects. A placebo-controlled, double-blind crossover study was undertaken in 10 normal subjects to examine the effects of arotinolol (10 mg bid), a nonselective beta blocker with alpha-blocking activity, on exercise capacity and hormone levels during exercise after a 2-week treatment period. Maximal oxygen uptake (VO2 max) and blood lactic acid concentration (LA) were measured during progressive exercise testing. An exercise intensity equivalent to 4 mmol/l of LA was used for the constant workload exercise test. Humoral factors were measured after 20 minutes of constant workload exercise. The administration of arotinolol significantly decreased systolic blood pressure and heart rate at rest and during exercise, but diastolic blood pressure did not change. No significant difference was found between arotinolol and placebo with regard to VO2 max and maximal workload. Plasma renin activity (PRA), aldosterone (PAC), and norepinephrine (NE) levels at rest and during exercise did not differ between the two treatments. In contrast, plasma epinephrine (EN) levels at rest and during exercise were significantly greater with arotinolol. Atrial natriuretic peptide (ANP) at rest did not differ between the two treatments. However, exercise caused a significant increase in ANP after arotinolol treatment. These findings suggest that arotinolol decreases blood pressure and heart rate without affecting exercise capacity.


Quantitation of physical-chemical properties of the aqueous phase inside the phoE ionic channel. The anion-specific channel of the phoE porine is a miniature body of water surrounded by peptide walls. The physical and chemical properties of the water in such a microscopic space were measured by monitoring the dynamics of a well-studied reaction--the protolytic dissociation of a strong acid. To attain this purpose, we allowed pyranine (8-hydroxypyrene-1,3,6-trisulfonate) to bind to the anion-specific channel. The dye is bound, with a 1:1 stoichiometry, with a delta G = -9.5 kcal/mol. Photoexcitation of the dye, to its first electronic singlet state (phi OH*), renders it very acidic and the hydroxyl proton dissociates to H+ and excited anion (phi O*-). We employed single photon-counting time-resolved fluorimetry, to monitor the reversible dissociation of pyranine as it proceeds within the channel and reconstructed the observed signal by a numerical integration of the differential diffusion equation pertinent for a proton within the channel. The most characteristic feature of the water-filled channel, is the intensified electrostatic interactions attained by the low dielectric constant of the diffusion space, epsilon eff = 24. For this reason, the electric field of a few positive charges is sufficient to ensure that an anion entering the channel will be effectively sucked in. The interaction of the water molecules with the peptide structure forming the channel affects the physical properties of the water. Their capacity to conduct proton, quantitated by the protons diffusion coefficient (4.5.10(-5) cm2/s), is reduced by 50% with respect to that of bulk water. The activity of the water in the channel is reduced to alpha H2O = 0.966. These observation are in accord with our previous studies of water in small defined cavities in proteins.


Cholecystokinin-induced changes in polysome structure regulate protein synthesis in pancreas. Translational regulation of digestive enzyme synthesis during short-term stimulation by cholecystokinin-octapeptide (CCK-OP), was examined in minced rabbit pancreas by measuring protein synthesis and monitoring alterations in the size of polysomes attached to the rough endoplasmic reticulum (RER). The effect of CCK-OP on protein synthesis was determined by measuring [3H]leucine incorporation into trichloroacetic-acid-precipitable proteins. Concentrations of CCK-OP that caused maximal enzyme secretion (10 and 30 nM) decreased protein synthesis by approx. 50% compared to control. Protein synthesis returned to the control level 60 min after terminating the action of CCK-OP. Autoradiography of [35S]methionine-labeled proteins separated by one-dimensional SDS-polyacrylamide gel electrophoresis demonstrated that CCK-OP reversibly inhibited the synthesis of all of the major groups of digestive enzymes. Northern blot analysis revealed that CCK-OP did not alter the cellular content of amylase and elastase mRNA. Incubation with CCK-OP caused a decrease in the size distribution of RER-bound polysomes. Polysome profiles returned to the control pattern 60 min following termination of the stimulus. These results suggest that the inhibitory effects of CCK-OP on the synthesis of digestive enzymes is regulated at translation by decreasing the number of RER-bound ribosomes that are actively translating digestive enzyme mRNA.


Differential regulation of glucose transporter isoforms by thyroid hormone in rat heart. Since thyroid hormone stimulates cardiac metabolism, for which glucose is an important fuel, we examined the effects of thyroid hormone on glucose transporter gene expression in rat heart. Treatment of hypothyroid animals with T3 for up to 6 days caused a 2-fold increase in GLUT4 mRNA at 1 day, and a 4-fold increase at 6 days (per unit DNA). GLUT4 protein, however, was not increased. In contrast, GLUT1 mRNA was transiently decreased by T3 treatment at 1 and 3 days, but returned to normal by 6 days. Concomitantly, GLUT1 protein decreased to 18% of the control value at 6 days. In chronically hypothyroid or hyperthyroid rats, GLUT4 mRNA varied directly with thyroid status, but GLUT4 protein was invariant, consistent with the acute effects of T3 treatment. Moreover, hypothyroidism increased GLUT1 mRNA and protein expression. We conclude that, in contrast to previous observations in skeletal muscle, GLUT4 protein content in rat heart is not altered by thyroid hormone. Cardiac GLUT1 expression is increased in hypothyroidism, and suppressed by thyroid hormone, at both the protein and mRNA levels. The observed discrepancy between GLUT4 mRNA and protein levels suggests that post-transcriptional regulatory events play a major role in GLUT4 expression in rat heart.


Kinetics and stability of delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni in the system of reverse micelles of aerosol OT in isooctane. Partially purified delta 5-3-ketosteroid isomerase (KSI) from Pseudomonas testosteroni was studied kinetically after solubilization in reverse micelles of aerosol OT (AOT) in isooctane and water, as regards its application to biotechnology. With delta 5,10-estren-17 beta-ol-3-one as a substrate, KSI displays an enzyme activity in the micellar system but a low stability. In the presence of urea, the enzyme is, however, stable. Kinetic parameters of the stabilized enzyme are highly sensitive to both the hydration degree of the surfactant and its concentration. The hypothesis of the geometric correspondence of a non-spherical enzyme and spherical micellar matrix is considered.


The 23S-5S spacer of two rRNA loci of Streptococcus salivarius subsp thermophilus includes a promoter. The nucleotide sequence of the 3' part of a ribosomal and transfer RNA locus from Streptococcus salivarius subsp thermophilus NST1403 was determined. The sequenced DNA fragment includes the 3' end of a 23S rRNA gene, a 5S rRNA gene, a tRNA(asn) gene and a potential transcriptional terminator. The tRNA gene does not encode for the CCA 3'terminus of mature tRNA. We compared this sequence to a promoter-carrying DNA fragment sequence (P20) of Streptococcus salivarius subsp thermophilus A054 [1]. We found that the P20 sequence included the 3' end of a 23S rRNA gene, a 5S rRNA gene and the 5' part of a tRNA(val) gene. The two 23S-5S spacer sequences are identical and contain a promoter and a potential 23S rRNA processing site. Therefore, 5S rRNA and tRNA genes could be transcribed from a promoter located within the 23S-5S spacer of at least two of the six rRNA loci.


Identification and epidemiological typing of clinical and environmental isolates of the genus Rhodococcus with use of a digoxigenin-labeled rDNA gene probe. Rhodococcus species are ubiquitous in the environment, and several species have been reported to have pathogenic potential for humans. Rhodococcus equi, in particular, has been reported to cause infections in patients with AIDS. However, the identification of Rhodococcus species with use of conventional biochemical tests is problematic, and no simple and reproducible method exists for their rapid identification and differentiation. We found that the type strains of the 20 recognized species in the genus Rhodococcus could be clearly distinguished with use of a combination of the Pvu II and Pst I rRNA gene restriction endonuclease patterns and a digoxigenin-labeled Escherichia coli rDNA probe. Analysis of four clinical or environmental isolates confirmed as Rhodococcus bronchialis showed no interstrain variation of rRNA gene bands. Analysis of 15 isolates confirmed as R. equi from 13 patients showed 11 different rRNA gene patterns. No discernible difference was observed in the ribotype patterns between R. equi isolates from patients for whom AIDS had been diagnosed and those from patients who did not have AIDS, and there was no evidence of geographic clustering of R. equi ribotype patterns. Three of five Rhodococcus species isolates that could not be differentiated with use of conventional biochemical methods were identified with use of ribotype analysis. Therefore, ribotype analysis may provide an important adjunct to current biochemical identification of environmental and clinical isolates of Rhodococcus species.


[Photochemical transformation of Hoescht 33258 dye molecules complexed with cell nucleus DNA under the effect of UV-irradiation]. It is found that with time a decrease of fluorescence intensity of the basic band at 460 nm and appearance of a new band of fluorescence of DNA-specific dye Hoechst 33258 in complex with the cell nucleus DNA under the action of UV emission are observed. It is shown that phototransformation is related to the withdrawal of the nitrogen atom proton of piperazine ring in an excited state of the complex of the dye Hoechst 33258 with the cell nucleus DNA.


Hypothesis for vaccine development: protective immunity to enteric diseases caused by nontyphoidal salmonellae and shigellae may be conferred by serum IgG antibodies to the O-specific polysaccharide of their lipopolysaccharides. Immunoprophylaxis for bacterial enteric diseases is hindered because the protective immune mechanism(s) against nontyphoidal salmonellae or shigellae in humans are not established. On the basis of the similarities between the clinical signs, epidemiology, pathogenesis, and pathology of as well as protective immunity to salmonellae and shigellae, we propose that serum IgG antibodies to the O-specific polysaccharide (O-SP) of their lipopolysaccharides (LPSs) will confer protective immunity to these two pathogens. Critical to this notion is that (1) the virulence of these two pathogens requires full expression of their LPS; (2) active or passive immunization with serum IgG O-SP antibodies confers protection of mice against Salmonella typhimurium (there are no comparable data for humans); and (3) in humans, convalescence from shigellosis confers type (O-SP) -specific protective immunity, and indirect evidence shows a correlation between the level of serum LPS antibodies and resistance to shigellosis. We designed conjugate vaccines to elicit high levels of long-lived serum IgG O-SP antibodies to nontyphoidal salmonellae and shigellae to test this hypothesis.


[Endothelium-derived relaxation factor from vessels--a nitrosyl iron complex with thiol-containing ligands (hypothesis)]. A hypothesis is presented concerning the nature of endothelium--derived relaxing factor (EDRF) of blood vessels--a compound isolated from endothelial cells under the effect of acetylcholine, bradykinin and other agents. This factor causes relaxation of the smooth muscles of these vessels. EDRF is suggested to be a nitrosyl complex of iron with low-molecular thiol-containing ligands, most probably with cysteine. Nitric oxide produced from arginine by NO-synthetase is their active component. Incorporation of NO into iron complexes stabilizes it providing transfer inside the cells and between them. Subsequent liberation of NO from these complexes results from the oxidation of thiol ligands.


Construction of protein analogues by site-specific condensation of unprotected fragments. The extreme sensitivity to periodate of 1-amino, 2-hydroxy compounds permits the selective conversion of N-terminal serine and threonine to an aldehydic group. We have used this reaction to construct analogues of human granulocyte colony stimulating factor (G-CSF) by allowing such oxidized peptides to react with others that have had a hydrazide derivative attached to the C-terminus by reversed proteolysis. Two recombinant analogues of G-CSF were used as starting materials. Both had only a single lysine residue (at position 62 and 75, respectively) followed immediately by a serine. Digestion of each analogue by the lysine-specific protease from Achromobacter lyticus gave two fragments, one of which could be N-terminally oxidized and the other converted to the C-terminal hydrazide derivative by reversed proteolysis using the same enzyme. After preliminary studies with model peptides, we first reacted the corresponding peptide pairs together and then, in order to eliminate the 64-74 disulfide loop, fragment 1-62 from the first analogue with fragment 76-174 from the second. Reactions are efficient (up to 80% product based on the oxidized fragment) and take place under very mild conditions. The hydrazone bond can easily be stabilized by reduction with NaBH3CN. This method represents a new, reasonably general route for the construction of large protein chimeras of precisely controlled structure.


Ex vivo expansion and maturation of peripheral blood CD34+ cells into the myeloid lineage. Hematopoietic reconstitution (HR) after peripheral blood stem cell transplantation is characterized by a delay of 8 and 12 days for recovery to safe levels of neutrophils and platelets even in patients with the most rapid engraftment. We postulate that a further enhancement in the rate of HR may be achieved by transplanting with an expanded postprogenitor cell population that can provide mature functional cells within days of infusion. In this study we investigated the ability of combinations of hematopoietic growth factors (HGF) to generate nascent granulocyte-macrophage colony-forming units (CFU-GM) in a 7-day suspension culture of peripheral blood CD34+ cells. A combination of 6 HGF, ie, interleukin-1 beta (IL-1), IL-3, IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), and stem cell factor (SCF), was identified as the most potent combination of those tested. Subsequently, large volume suspension cultures of CD34+ cells from the same patients using the same 6-factor combination were established and monitored for 21 days. An exponential rate of nucleated cell production (mean 1,324-fold increase) occurred during culture. CFU-GM production paralleled nucleated cell production until day 10, peaked at day 14 (mean 66-fold increase), and was then maintained until day 21. Cells produced in culture were predominantly neutrophil precursors and developed normally as assessed by morphology, immunophenotype, and superoxide generation. This stroma-free, cytokine-driven culture system can achieve a degree of amplification, which suggests the feasibility of ex vivo culture of hematopoietic progenitor cells as an adjunct to hematopoietic stem cell transplantation.


Transcripts of the mitochondrial gene ND5 are overexpressed in highly metastatic murine large cell lymphoma cells. The relative levels of mitochondrial specific gene transcripts were compared in two murine large cell lymphoma cell lines that differ in their propensities to form liver metastases and in their sensitivity to macrophage mediated antitumor cytostasis and cytolysis. Full-length transcripts of the mitochondrial genes were hybridized on electroblots from citrate/urea gels with specific gene prodes. The mitochondrially encoded gene NADH dehydrogenase subunit 5 (ND5), that encodes a component of NADH dehydrogenase (complex I) of the electron transport chain, was significantly overexpressed in the highly metastatic RAW117-H10 compared to low metastatic RAW117-P cells. Results from analysis of RNA blots were confirmed in an S1 nuclease protection assay. Since RAW117-H10 cells are significantly more resistant to cytostasis by activated macrophages in coculture and such macrophage activity can inhibit RAW117 tumor cell respiration and growth, a mechanism was suggested that allows RAW117 cell escape from certain host effector mechanisms that block cellular respiration by an increase in the in vivo concentrations of translatable messenger RNA (mRNA) that codes for key components of the electron transport chain.


Role of the modified (glycosaminoglycan producing) perisinusoidal fibroblasts in the CCl4-induced fibrosis of the rat liver. Development and regression of liver fibrosis and cirrhosis induced by CCl4 in male F-344 rats were strictly followed during and after an 8-week treatment. The relative amount of collagen was measured by morphometry and the number of glycosaminoglycan (GAG) containing fat storing cells was counted at each time point. The expression of proteoglycan genes (decorin, versican and BPG-5 HSPG) was studied in parallel with the development of cirrhosis. Collagen content of the liver as well as the number of GAG-containing mesenchymal (fat storing) cells increased in parallel until two weeks after the cessation of CCl4 treatment. Later, both the collagen content and the number of GAG-containing cells decreased in parallel and significantly. Proteoglycan gene expression in the nonparenchymal fraction of liver cells indicated an active proteoglycan synthesis in the course of the development of cirrhosis. It is concluded that modified Ito (fat storing) cells synthesize proteoglycans and play an important role in the formation of connective tissue fibers in liver fibrosis.


Granulocyte colony-stimulating factor to prevent dose-limiting neutropenia in non-Hodgkin's lymphoma: a randomized controlled trial. The effect of granulocyte colony-stimulating factor (G-CSF) on neutropenia, infection, and cytotoxic chemotherapy administration was studied in a randomized trial in patients receiving intensive weekly chemotherapy for non-Hodgkin's lymphoma (NHL). Eighty patients (aged 16 to 71 years) with high-grade NHL (Kiel) of any stage were randomized to receive VAPEC-B chemotherapy alone (39 patients) or with G-CSF administered as a daily subcutaneous dose of 230 micrograms/m2 (41 patients). Prophylactic ketoconazole and cotrimoxazole were administered to all patients throughout treatment. The protocol specified identical dose modification and antibiotic treatment criteria bor both groups. Neutropenia (absolute neutrophil count [ANC] less than 1.0 x 10(9)/L) occurred in 15 of 41 (37%) of the G-CSF-treated patients and in 33 of 39 (85%) of the controls, giving a relative risk for control patients of 2.31 (95% confidence interval [CI], [1.51, 3.54]; P = .00001). Fever (greater than or equal to 37.5 degrees C) with neutropenia (ANC less than 1.0 x 10(9)/L) occurred in 9 of 41 (22%) of the G-CSF group and in 17 of 39 (44%) of the controls (relative risk for control, 2.26; 95% CI [1.01, 5.06]; P = .04). There were fewer treatment delays, with shorter duration (P = .01) in patients receiving G-CSF. Chemotherapy doses were reduced in 4 of 41 (10%) of the G-CSF patients and 13 of 39 (33%) of the controls (P = .01). The dose intensity of cytotoxic chemotherapy was significantly increased in patients receiving G-CSF (median of 95% in G-CSF group compared with 83% in control patients). Three vascular deaths occurred in the G-CSF group. Delays in the control group most commonly resulted from neutropenia (19 patients, compared with 2 patients in the G-CSF-treated group, P = .000007). Severe mucositis was the major dose-limiting toxicity in G-CSF-treated patients, but did not occur more frequently than in controls (15 patients in each group). Overall, patients randomized to receive G-CSF achieved a greater dose intensity than control patients, but this did not result in significant differences in drug toxicity (other than neutropenia), intravenous antibiotic usage, or hospitalization between the two groups.


c-kit Gene was not transcribed in cultured mast cells of mast cell-deficient Wsh/Wsh mice that have a normal number of erythrocytes and a normal c-kit coding region. The Wsh is a mutant allele at the W (c-kit) locus of mice. Mice of Wsh/Wsh genotype have white hairs and black eyes. Although adult C57BL/6-Wsh/Wsh mice were not anemic, they showed a remarkable depletion of mast cells. Most homozygous or double heterozygous mutant mice at the W (c-kit) locus, of which mast-cell depletion was comparable to that of Wsh/Wsh mice, are deficient in germ cells. However, male and female Wsh/Wsh mice have an appreciable number of germ cells in their gonads. We investigated the mechanism of specific depletion of mast cells in Wsh/Wsh mice. Cultured mast cells (CMC) derived from the spleen of Wsh/Wsh mice neither attached to normal (+/+) fibroblasts nor survived in the coculture with +/+ fibroblasts. The c-kit messenger RNA (mRNA) was strongly expressed in +/+ CMC, but not detectable in Wsh/Wsh CMC. Despite the lack of c-kit mRNA in Wsh/Wsh CMC, the c-kit mRNA was normally detectable in the cerebellum and weakly detectable in the testis and spleen of Wsh/Wsh mice. No significant changes were found in the nucleotide sequence of the c-kit transcripts obtained from the cerebellum of Wsh/Wsh mice. Development of mast cells, erythrocytes, and germ cells in Wsh/Wsh mice appeared to be parallel with the magnitude of the c-kit gene expression in each cell type.


Low c-kit expression of cultured mast cells of mi/mi genotype may be involved in their defective responses to fibroblasts that express the ligand for c-kit. Mutant mice of mi/mi genotype are osteopetrotic and deficient in tissue mast cells due to a defect in osteoclasts and mast cells. In an effort to further understand the mechanisms behind why mi/mi mouse-derived cultured mast cells (mi/mi-CMC) responded to interleukin-3 (IL-3), but not to the proliferative stimuli presented by fibroblasts, mi/mi-CMC and congenic normal (+/+) mouse-derived CMC (+/+-CMC), both of which expressed the phenotypic characteristics of immature mast cells, were cocultured with Swiss albino/3T3 fibroblasts in a medium containing IL-3. In the in vitro CMC/fibroblast coculture, mi/mi-CMC did not acquire the phenotypes of connective tissue-type mast cells (CTMC), while +/+-CMC did. In addition, attachment of mi/mi-CMC to the fibroblasts was found to be significantly lower than that of +/+-CMC. Because the interaction of c-kit product with its ligand (stem cell factor [SCF]) is known to play an important role not only in proliferation and differentiation of mast cells but also in attachment of CMC to fibroblasts, the expression and function of c-kit were investigated in mi/mi-CMC and +/+-CMC. Recombinant rat SCF (rrSCF164) induced a dose-dependent proliferation of +/+-CMC. Also, rrSCF164 induced +/+-CMC to acquire the phenotypes of CTMC in the medium containing IL-3. By contrast, rrSCF164 did not stimulate the proliferation of mi/mi-CMC nor induce a phenotypic change of the cells from immature mast cells to mature, CTMC-like mast cells. Immunoblotting with antiphosphotyrosine antibody showed that rrSCF164 induced considerable tyrosine phosphorylation of 145- to 165-Kd protein, the product of c-kit, in +/+-CMC, whereas tyrosine phosphorylation of the protein was barely detectable in mi/mi-CMC. Northern blot and flow cytometry analyses showed that mi/mi-CMC expressed much less c-kit at both protein and message levels than +/+-CMC. Further, mi/mi-CMC were found to differ from +/+-CMC in the expression of mouse mast cell protease-6 (MMCP-6) and MMCP-2 messenger RNA transcripts. These results suggest that the gene product of the mi locus may be important in regulating the expression of gene products such as c-kit, and that mast cell deficiency of mi/mi mice appears to be due, at least in part, to impaired signaling through the c-kit receptor because of the low c-kit expression.


Immunosuppressants FK506 and rapamycin function as reversal agents of the multidrug resistance phenotype. The multidrug-resistant (MDR) phenotype is characterized in vitro by the resistance displayed by cell lines to a broad spectrum of natural product cytotoxic agents. This high level of cross-resistance is due to the increased expression of a membrane glycoprotein termed P-glycoprotein. Encoded in humans by the mdr1 gene, P-glycoprotein functions as an energy-dependent efflux pump of these cytotoxic agents. In this report, we demonstrate that the newly characterized immunosuppressant FK506 and its structural analogue, rapamycin, are capable of functioning as MDR reversal agents. FK506 and rapamycin increase both intracellular, cytotoxic drug (daunomycin) accumulation, and the cytotoxicity of chemotherapeutic agents in multidrug-resistant cells. The increase in cytotoxic drug accumulation is observed at concentrations of FK506 and rapamycin 1,000-fold greater than the concentrations required for FK506 and rapamycin to inhibit T-lymphocyte activation and similar to those shown to be effective for other MDR reversal agents such as cyclosporine A (CsA) and verapamil. The effect of FK506 or rapamycin on both intracellular accumulation and cytotoxicity of daunomycin is additive. This is supported by the ability of FK506 and rapamycin to directly compete the binding of the photoaffinity analogue 125I-iodoaryl azidoprazosin to the P-glycoprotein. The data demonstrate that FK506 and rapamycin represent a new class of structurally distinct molecules that can function as MDR reversal agents and suggest a previously unidentified, potential clinical role for these compounds.


Induction of erythroid differentiation and fetal hemoglobin production in human leukemic cells treated with phenylacetate. There is considerable interest in identifying nontoxic differentiation inducers for the treatment of various malignant and nonmalignant blood disorders, including inborn beta-chain hemoglobinopathies. Using the human leukemic K562 cell line as a model, we explored the efficacy of phenylacetate, an amino acid derivative with a low toxicity index when administered to humans. Treatment of K562 cultures with pharmacologically attainable concentrations of phenylacetate resulted in erythroid differentiation, evident by the reduced growth rate and increased hemoglobin production. The effect was time- and dose-dependent, further augmented by glutamine starvation (phenylacetate is known to deplete circulating glutamine in vivo), and reversible upon cessation of treatment. Molecular analysis showed that phenylacetate induced gamma globin gene expression with subsequent accumulation of the fetal form of hemoglobin (HbF). Interestingly, the addition of phenylacetate to antitumor agents of clinical interest, eg, hydroxyurea and 5-azacytidine, caused superinduction of HbF biosynthesis. The results suggest that phenylacetate, used alone or in combination with other drugs, might offer a safe and effective new approach to treatment of some hematopoietic neoplasms and severe hemoglobinopathies.


Microbiologic and pathologic aspects of pulpal and periapical disease. The greatest cause of endodontic and periapical pathosis is microbial infection of the pulp. Most odontogenic infections are of a polymicrobial nature. With advances in anaerobic isolation and culturing techniques, much has been learned about the presence of pathogenic organisms such as Porphyromonas and Prevotella species (formerly classified as black-pigmented Bacteroides species) in infected root canals. This review provides a summary of recent developments in endodontic microbiology, virulence factors, and host defense systems as they relate to the pathogenesis of pulpal and periapical inflammatory lesions.


Teicoplanin in open fractures: a preliminary report. The occurrence of infection following open fractures varies with the severity of the fracture and associated soft tissue damage. In order to study the effect of antibiotics given prophylactically, teicoplanin has been used in a prospective study. On admission, fractures were graded according to the severity of the tissue damage: Type I--soft tissue injury less than 1 cm Type II--soft tissue injury more than 1 cm. Type III fractures characterized by extensive soft tissue and bone damage were not included in the study. Type I patients were given a single dose of teicoplanin, 400 mg i.v.; Type II patients were given a single dose of teicoplanin, 400 mg i.v., followed by two further intravenous injections at 12 and 24 hours. Patients were assessed for the presence of wound and bone infections and follow up was for 1 year. Preliminary results, predominantly from Type II fractures involving long bones, show that teicoplanin is effective in lowering the incidence of infection following open fractures.


Teicoplanin vs cephamandole for antimicrobial prophylaxis in prosthetic joint implant surgery: (preliminary results). Infection following prosthetic joint implant jeopardizes the prosthesis and may lead to long-term locomotor disability. Interoperative antimicrobial prophylaxis can reduce the incidence of infectious complications. The comparative safety and efficacy of single-dose teicoplanin and four doses of cephamandole over a 24-hour period is currently being assessed in a single-blind, randomized concurrent study of patients who undergo first time hip or knee arthroplasty. Of 660 evaluated patients, 352 have received cephamandole and 308 teicoplanin. Two patients in each group had a surgical wound infection at 1 week after surgery. Reassessment 30 days postoperatively showed resolution of the infection in both of the teicoplanin patients and in one of the cephamandole patients. Proven or suspected infection involving other body systems occurred in 30 teicoplanin and 38 cephamandole patients at 1 week postoperatively and in 5 teicoplanin and 3 cephamandole patients 1 month after surgery. Adverse events occurred in 20 (5.1%) teicoplanin patients and 29 (7.1%) cephamandole patients. These preliminary results suggest that single-dose teicoplanin is a safe and effective prophylactic agent in prosthetic joint implant surgery.


Teicoplanin--home therapy for prosthetic joint infections. Infection with methicillin-resistant organisms is increasingly common among implanted orthopaedic devices. The organisms involved are often multiresistant to other commonly used antibiotics. Rifampicin resistance is less common at isolation but may develop during treatment unless combination therapy with another drug is employed. However, tolerance of combinations is poor, particularly among elderly patients. These patients may require at least 6 weeks of antibiotic therapy, and where there is no suitable oral therapy, this has meant prolonged hospitalization solely for administration of parenteral antibiotics. Recently we have started treating such patients with teicoplanin, 6 mg/kg once daily, by intravenous bolus injection. The injections are administered by relatives at home and the patients attend the ward twice weekly for inspection and changing of the intravenous cannula. The potential for home administration of parenteral teicoplanin gives this drug a major advantage over other parenteral drugs for the treatment of prosthetic joint infections.


Teicoplanin--domiciliary use in surgical infections. This is an ongoing study designed to evaluate the role of teicoplanin in the therapy of sternal wound infections following cardiac surgery caused by Gram-positive microorganisms. Intravenous teicoplanin therapy is begun in the hospital, but once the patients return home it is given as a single intramuscular dose of 400 mg/day. Only a limited number of patients have been recruited so far, but in these, the clinical success rate and the bacteriological elimination rate are 85%. Only one patient had to be withdrawn from the study due to intolerable side-effects. These preliminary results suggest that teicoplanin is suitable for the therapy of chest wound infections following cardiac surgery; a major part of the course can be administered at home in a single daily intramuscular injection. This novel therapy regimen is expected to reduce considerably the hospital costs involved in the therapy of sternal wound infections.


Teicoplanin--its role as systemic therapy of burn infections and as prophylaxis for orthopaedic surgery. Italian Study Groups for Antimicrobial Prophylaxis in Orthopaedic Surgery and Burns. Two randomized studies have been initiated to establish the role of teicoplanin as systemic therapy for infections in burns patients and as short-term prophylaxis for orthopaedic implant surgery. Opportunistic micro-organisms causing infections in burn patients are often acquired in hospital. These infections commonly involve Gram-positive organisms which may be resistant to several antibiotics. Teicoplanin, alone and in combination with additional antibacterial drugs, is effective in the treatment of Gram-positive infections of various types. In addition, teicoplanin has proved useful as prophylaxis against infection in orthopaedic surgery. Deep prosthetic infections are very difficult to cure without removing the infected device; the outcome can be devastating, such as total loss of joint function, amputation, and, occasionally, death. Preliminary results from the two studies are encouraging and show that teicoplanin has a role to play both in treatment of infection and as prophylaxis against hospital-acquired infection.


Antibiotic prophylaxis in clean surgical cases and the role of community surveillance. In an ongoing prospective study of clean surgical procedures, patients have been examined by specialist research nurses on a minimum of three occasions during the postoperative period to determine the incidence of wound complications. Whereas a previous retrospective audit suggested a wound infection rate of 2%, this community surveillance programme revealed the true rate as at least fourfold greater (p less than 0.001). Of these complications, more than 70% were detected only by the surveillance programme. Immediate benefit was obtained by a reduction in all complications. Despite these improvements, a core of postoperative sepsis remained, particularly in high-risk cases. Thus, a prospective, controlled, prophylactic antibiotic trial has been undertaken, in an attempt to reduce the infection rate still further. An antibiotic regimen of teicoplanin, 400 mg i.v., given as a single dose, was chosen because of the excellent activity of this agent against staphylococci and streptococci, and also because of the long half-life which renders it suitable for single-dose administration.


Efficacy, tolerability and pharmacokinetics of teicoplanin in patients undergoing breast surgery. In a prospective open trial, 21 female patients undergoing breast surgery received a single intravenous bolus injection of teicoplanin, 400 mg, 30 minutes before the operation. During surgery, (1 hour after teicoplanin administration), samples of breast and fat tissue and serum were collected; 24 hours and 48 hours after dosing, samples of wound exudate were taken. Teicoplanin concentrations were determined using the RASA method. Teicoplanin levels were lowest in fat tissue. Teicoplanin levels in wound exudate were found to be satisfactorily high. However, teicoplanin levels in fat and breast tissue and in wound exudate exceeded the MIC90 for various staphylococcus strains, such as S. aureus and S. epidermidis. No local or systemic adverse reactions were observed during the trial. Teicoplanin was well tolerated. Preliminary results from this trial, therefore, indicate that teicoplanin may be a suitable antibiotic for use in the prophylaxis of infection in breast surgery.


Teicoplanin and prophylaxis of Hickman catheter insertions. A total of 88 patients with haematological malignancies who required Hickman catheters for intensive chemotherapy, were randomized to receive either a single bolus intravenous injection of teicoplanin, or no teicoplanin, immediately before insertion of a double lumen Hickman catheter. There was a lower incidence of catheter-related Gram-positive sepsis in patients receiving prophylactic teicoplanin. This benefit was particularly clear in patients who were already neutropenic at the time of catheter insertion. Prophylactic teicoplanin may, therefore, be useful as a routine procedure during the insertion of Hickman catheters for this group of patients.


Teicoplanin vs cephradine and metronidazole in the prophylaxis of sepsis following vascular surgery: an interim analysis of an ongoing trial. This paper presents further preliminary results of a trial of the prophylaxis of sepsis in 165 patients undergoing vascular surgery. The efficacy and safety of a single dose of teicoplanin was examined and compared with three doses of cephradine plus metronidazole. No significant differences were detected in the prophylactic efficacy in either group. The first interim report indicated abnormalities in liver function, maximum at 7 days, in both groups. These findings are confirmed in this second interim report. Raised levels of GGT and alkaline phosphatase are more prominent in patients receiving teicoplanin. Liver function improved by 28 days, however, suggesting that any abnormality is transient.


Teicoplanin in the treatment of bone and joint infections. Teicoplanin Bone and Joint Cooperative Study Group, USA. Teicoplanin is a new glycopeptide antibiotic with activity against Gram-positive bacteria, including methicillin-resistant organisms. Teicoplanin is administered once daily, either intravenously or intramuscularly. Teicoplanin was given once daily, intravenously or intramuscularly, in the treatment of hospitalized or ambulatory patients with Gram-positive bone or joint infections. A total of 90/98 patients were evaluated for efficacy; 41 had acute osteomyelitis, 41 had chronic osteomyelitis, and 8 had septic arthritis. At the end of therapy, 37 acute osteomyelitis patients were cured/improved with a 90% cure rate at 6-month follow-up; 2 relapsed and 1 failed. At the end of therapy 30 chronic osteomyelitis patients were cured/improved with an 88% cure rate at 6-month follow-up; 2 relapsed and 1 failed. 100% of the septic arthritis patients were cured at the end of therapy and at 1-month follow-up. The most common bacterial isolates cultured from bone were S. aureus (39 isolates), S. epidermidis (11 isolates), other coagulase-negative staphylococci (20 isolates), enterococci (6 isolates), and other streptococcal species (20 isolates). The most common bacterial isolates cultured from joint fluid were S. aureus (6 isolates) and S. epidermidis (2 isolates). All patients tolerated the intramuscular or intravenous routes of administration well. Adverse reactions were mild and most cases did not require discontinuation of therapy. The majority of therapy was administered on an outpatient basis. Teicoplanin was safe, effective, convenient and relatively well tolerated in patients with acute or chronic osteomyelitis or septic arthritis.


Vitronectin-binding surface proteins of Staphylococcus aureus. S. aureus strain ISP 546 was selected (of 55 strains tested) to define optimal conditions for expression of vitronectin binding. High binding was expressed when the strain was grown on blood agar and in Todd-Hewitt broth. Binding was optimal in the 6.0 to 7.2 pH range and was unaffected by divalent cations and ionic strength. Binding was partially inhibited by D-mannose, heparin, types I and IV collagen, fibronectin, fibrinogen and vitronectin, but was not affected by other carbohydrates or glycoproteins tested. Cell surface binding components were extracted with the aid of 1 M LiCl (pH 5.0) from strain ISP 546 grown in Todd Hewitt broth. Vitronectin binding proteins were purified by affinity chromatography on heparin-Sepharose. Fractions inhibiting binding of 125I-labelled vitronectin to strain ISP 546 were eluted by 0.01 M NaOH, dialysed, concentrated and subjected to SDS-PAGE. Silver staining revealed one major band (70 kDa) and two minor bands (34 and 36 kDa).


Binding of laminin, type IV collagen, and vitronectin by Streptococcus pneumoniae. Forty-three strains of Streptococcus pneumoniae were tested for their ability to bind radiolabelled laminin, collagen types I, II and IV, fibronectin, and vitronectin. Two basement membrane components, laminin and type IV collagen, interacted with many S. pneumoniae strains. All strains bound laminin and 28 (65%) bound collagen type IV. Approximately 60% of the strains bound vitronectin but only a few strains showed low binding of fibronectin and collagen type I and II.


Some biochemical characteristics of a partially purified extracellular keratinase from Trichophyton schoenleinii. A Trichophyton schoenleinii (T. schoenleinii) strain from tinea favus was cultured in a liquid medium, from which an extracellular keratinase extract was obtained. The keratinase was partially purified with carboxymethyl-cellulose (CMC) column chromatography. Some biochemical characteristics of the keratinase were then examined. Its molecular weight was estimated to be 38,000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal temperature was 50 degrees C, and the optimal pH value was 5.5. The keratinolytic activity was specifically increased by Fe++ and Fe .


Axonal blockade induces the expression of vasoactive intestinal polypeptide and galanin in rat dorsal root ganglion neurons. Nerve growth factor (NGF) undergoes retrograde transport from peripheral target organs, and has been recently reported to regulate the production of some neuropeptides in dorsal root ganglion (DRG) neurons. Therefore, to ascertain whether or not the expression of calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), and galanin was regulated by the retrograde transport of factors such as NGF, we carried out an immunocytochemical analysis using vinblastine as an axonal transport blocker and a monoclonal antibody to the NGF receptor (NGFR) as a marker of NGF-responsive neurons. The percentage of CGRP-containing DRG neurons (L5) was decreased by sciatic nerve transection or by the application of higher doses of vinblastine (0.3-0.6 mM) to the sciatic nerve. VIP and galanin were expressed in some DRG neurons after the application of a low dose of vinblastine (0.15 mM), which can block axonal flow without causing neuronal damage. The expression of these peptides was not affected by dorsal rhizotomy. About 70% of the CGRP-containing neurons also expressed NGFR, while most of the VIP-containing or galanin-containing neurons lacked NGFR. These findings indicate that the depletion of peripheral target-derived neurotrophic factor(s) other than NGF by axonal blockade may induce the gene expression of VIP and galanin.


Origins and collateralization of corticospinal, corticopontine, corticorubral and corticostriatal tracts: a multiple retrograde fluorescent tracing study. Cerebral cells of origin for the corticospinal (CST), corticopontine (CP), corticorubral (CR) and corticostriatal (CS) fibers in the rat were identified following the simultaneous retrograde transport of propidium iodide (PI), fast blue (FB), fluorogold (FG) and diamidino yellow (DY). PI was injected into the contralateral C4 spinal cord segment while FB, FG and DY were injected into the ipsilateral medial pontine nuclei, red nucleus and striatum, respectively. Labeled pyramidal neurons projecting corticospinal axons were contralateral to injection in lamina V and ranged in size from small to large. These CST neurons occupied two distinct cortical areas. The cortical neurons of origin for the corticopontine, corticorubral and corticostriatal fibers were ipsilateral to injections. Labeled neurons were localized in cortical lamina V for the corticopontine and corticorubral fibers while corticostriate neurons were located in laminae III, V and VI. The CP, CR and CS labeled cells occupied one large cortical area which topographically included parts of the medial (AGm) and lateral (AGl) agranular cortices and the primary (SI) somatosensory cortex. Considerable overlapping of the cortical neurons of origin for the four motor fiber systems was apparent. More than 98% of the labeled cells were single labeled while less than 2% were double labeled. No triple or quadruple labeled neurons were observed. Hence, morphological evidence is presented that cortical motor neurons project mainly individual, rather than collateral, axons to each of the four motor associated nuclei investigated in this study. However, only a few cortical neurons projected axons simultaneously to a maximum of two nuclei involved in the motor pathways.