Nucleotide sequence of a cDNA clone encoding a major allergenic protein in rice seeds. Homology of the deduced amino acid sequence with members of alpha-amylase/trypsin inhibitor family.

A cDNA clone of rice major allergenic protein (RAP) was isolated from a cDNA library of maturing rice seeds. The cDNA had an open reading frame (486 nucleotides) which coded a 162 amino acid residue polypeptide comprising a 27-residue signal peptide and a 135-residue mature protein of M(r) 14,764. The deduced amino acid sequence of RAP showed a considerable similarity to barley trypsin inhibitor [1983, J. Biol. Chem. 258, 7998-8003] and wheat alpha-amylase inhibitor [1981, Phytochemistry 20, 1781-1784].


Cyclic variation of major histocompatibility complex class II antigen expression in the human fallopian tube epithelium.

To systematically investigate the expression of major histocompatibility complex (MHC) class II antigens (human leukocyte antigens, HLA-DR, -DP, and -DQ) on columnar epithelium in the fallopian tube during the menstrual cycle. Biopsies were collected from the fallopian tube during laparotomy sterilization and immunoperoxidase staining was performed. Departments of Gynecology and Obstetrics and Clinical Immunology and Transfusion Medicine, Uppsala University, Uppsala, Sweden. Twenty healthy fertile women undergoing sterilization at different times of the menstrual cycle. None. The staining of the columnar epithelium was judged on a 4-graded scale according to the distribution of class II antigens. A widespread preovulatory HLA-DR expression was observed almost completely lining the columnar epithelial cells including the luminal surface, whereas postovulatory the HLA-DR expression was withdrawn from the surface. The HLA-DP and -DQ antigens varied in a similar way, although not as pronounced. The MHC class II antigen variation in the fallopian tube epithelium seen in this study may indicate a hormonal regulation that could reflect variable need for local immunocompetence during the menstrual cycle: a preovulatory need for immunoreactivity against invading microbes and postovulatory an optimal survival of the foreign preimplantation embryo.


Insulin-like growth factor I receptors in human corpora lutea.

To determine the presence and binding characteristics of insulin-like growth factor (IGF) receptors in corpora lutea (CL) of spontaneous and clomiphene citrate (CC)-induced cycles and to identify any relationship between IGF receptors and cytosol progesterone (P) and 17 alpha-hydroxyprogesterone (17 alpha-OHP) levels. Women undergoing bilateral tubal ligation were divided into two groups. One group received no medication (controls) and the other took 50 mg of CC. Midluteal phase CL were recovered at tubal ligation for hormone and receptor analysis. Patients were recruited from a university hospital setting. Eleven fertile women 26 to 37 years of age requesting bilateral tubal ligation were studied. Four women were given 50 mg/d of CC from days 5 through 9 of study cycle. Seven women did not take any medication. Minilaparotomy bilateral tubal ligation and luteectomy were performed 7 to 9 days after midcycle urinary (LH) surge. Insulin-like growth factor receptor concentrations and binding characteristics and cytosol P and 17 alpha-OHP levels in individual CL. Optimal binding for 125I-IGF-I with membrane fractions of homogenized CL was obtained with incubation at 4 degrees C for 16 hours. Specific binding (mean +/- SEM) was significantly higher in CC-treated (53.6% +/- 4.8%) than in control cycles (25.9% +/- 5.5%, P less than 0.001). Receptor concentrations were also significantly higher in CL from CC-induced (145.6 +/- 21.8 pmol/mg protein) than from control cycles (74.8 +/- 15.2 pmol/mg protein, P less than 0.02). Insulin-like growth factor receptor levels correlated with neither serum nor cytosol P and 17 alpha-OHP in CL from either cycles. Specific IGF-I receptors are present in human CL of the menstrual cycle with higher concentrations present in CC-induced cycles. Thus IGF may express its action on luteal function through its receptors in CL.


Testicular mast cell heterogeneity in idiopathic male infertility.

To determine if the mast cell subclass changes in diseased testes. Retrospective. University hospital urology clinic and anatomy laboratory. Fourteen normal men and 50 patients with idiopathic male infertility. Histochemical techniques to identify proteoglycans of mast cells were applied to the obtained testicular biopsy specimens. The numbers of the heparin containing mast cell and the chondroitin sulfate containing mast cell were measured with light microscopy. The total number of mast cells and the ratio of chondoroitin sulfate containing mast cells were significantly increased in the testes from patients with idiopathic male infertility. These results suggest that mast cells may play a certain role in the etiology of idiopathic male infertility.


The Steel factor.

Steel factor (SLF) is a recently identified growth factor which is the gene product of the murine Steel locus and a ligand for the c-kit tyrosine kinase receptor, the product of the dominant white spotting locus (W). Defects at these genetic loci result in aberrant melanocyte, germ cell, and hematopoietic development. Both the receptor (c-kit) and the ligand (SLF) have been shown to undergo tissue-specific mRNA splicing to produce distinct isoforms which have unique biological functions. As predicted by the phenotype of these mutations, SLF influences the growth and differentiation of melanocytes, primordial germ cells, and a broad spectrum of cell types in the hematopoietic progenitor and stem cell hierarchy. SLF has also been shown to have effects on hematopoietic lineages not predicted by defects seen in the Steel mouse.


A chondroitin sulfate proteoglycan that is developmentally regulated in the cerebellar mossy fiber system.

It is known that the mammalian brain contains many kinds of proteoglycans, but almost all of them remain to be characterized. In this study, we prepared a monoclonal antibody against a phosphate-buffered saline-soluble brain proteoglycan (MAb 6B4). MAb 6B4 recognized a 600- to 1000-kDa chondroitin sulfate proteoglycan with a 250-kDa core protein (6B4 proteoglycan). The core protein of 6B4 proteoglycan carried the HNK-1 epitope. Immunohistochemical analysis of the adult rat brain indicated that this proteoglycan was expressed on the cell surfaces of a subset of neurons. In the hindbrain, 6B4 proteoglycan was highly expressed on the cerebellar Purkinje cells and Golgi cells, and at particular nuclei including the pontine nuclei and lateral reticular nucleus. Almost all of these nuclei were connected to the cerebellum through the mossy fiber system. A developmental study indicated that the expression of this proteoglycan changed dramatically during the formation of the cerebellar mossy fiber system. The mossy fibers from the pontine nuclei expressed 6B4 proteoglycan transiently from Embryonic Day 20 (E20) to Postnatal Day 30 (P30), during which time the axonal outgrowth and glomerular synapse formation occurred. The Purkinje cells, glomeruli, and Golgi cells began to be stained with MAb 6B4 from P10, P16, and P20, respectively. These expression stages correspond with the onset of their synapse formation. These results suggest that 6B4 proteoglycan is closely involved in the development of the cerebellar mossy fiber system.


Insulin-like growth factor I and II and insulin-like growth factor binding protein-2 RNAs are expressed in adjacent tissues within rat embryonic and fetal limbs.

Since the rapid proliferation of cells in a directed manner is a necessary component of limb formation, the distribution of locally produced mitogenic molecules within the developing limb is of considerable interest. We have used in situ hybridization to localize transcripts for both insulin-like growth factor binding protein-2 (IGFBP-2) and its ligands, the insulin-like growth factors I and II (IGF-I and IGF-II), within limb buds of rat embryos 10-16 days after conception (equivalent to stages 1-12 of mouse limb morphogenesis, Wanek et al, 1989. J. Exp. Zool. 249, 41-49). The mRNA for IGFBP-2 is very abundant in an anterior-posterior strip of ectoderm along the distal edge of the limb bud (the progenitor of the apical ectodermal ridge or AER) from as early as limb stage 1 (Embryonic Day 10) and is much less abundant in the rest of the limb ectoderm. A high level of IGFBP-2 expression continues to characterize the AER following its definitive appearance (stage 3) and throughout its existence (until stage 7). This is a period of rapid outgrowth during which the rate of mesodermal cell division is highest in cells nearest to the AER. The AER is known to have mitogenic activity in vitro and to direct limb outgrowth in vivo, but, until recently, few putative molecular correlates of these activities have been detected. The transcripts for IGF-I and IGF-II are also present at high abundance in developing limbs, especially in mesodermally derived cells. IGF-I mRNA is abundant in presumptive limb mesoderm from the beginning of limb outgrowth (just before stage 1), but is very low or undetectable in much of the rest of the embryo, while IGF-II mRNA becomes very abundant in limb mesoderm at stage 2. The distribution in limbs of both IGF-I and IGF-II mRNA changes dramatically during outgrowth and differentiation, so that their expression characterizes complementary populations of cells by stage 11. Taken together, these data suggest that IGFs and the IGF binding proteins, which may modulate IGF action, contribute to limb outgrowth and patterning.


Enzymic activity of salivary amylase when bound to the surface of oral streptococci.

The enzymatic activity of salivary amylase bound to the surface of several species of oral streptococci was determined by the production of acid from starch and by the degradation of maltotetraose to glucose in a coupled, spectrophotometric assay. Most strains able to bind amylase exhibited functional enzyme on their surface and produced acid from the products of amylolytic degradation. These strains were unable to utilise starch in the absence of salivary amylase. Two strains failed to produce acid from starch, despite the presence of functional salivary amylase, because they could not utilise maltose. Strains that could not bind salivary amylase failed to produce acid from starch. In no case was all the bound salivary amylase active, and two strains of Streptococcus mitis which bound amylase did not exhibit any enzyme activity on their cell surface. The ability to bind amylase may confer a survival advantage on oral bacteria which inhabit hosts that consume diets containing starch.


The murine gene encoding the highly conserved Sm B protein contains a nonfunctional alternative 3' splice site.

The cDNA and partial genomic nucleotide (nt) sequences were derived for the mouse Sm B polypeptide and compared to the cDNA and genomic sequences encoding human Sm B. The deduced amino acid (aa) sequences from the mouse and human genes are identical with the exception of a single conserved aa substitution, accounting for the ability of anti-Sm antibodies to recognize the Sm polypeptides from a broad range of species. The genomic sequence of mouse B gene is similar to the human B genomic locus that extends from exon 6 to exon 7. These loci include conservation of both 3' alternative splice sites and putative branch points required to process B and B' mRNAs in human cells. However, the nt sequence downstream from the putative distal 3' splice junction and single nt flanking the 3' splice site consensus sequence, differ between mouse and human B. This results in a murine mRNA with a different predicted secondary structure around the distal 3' splice site when compared to humans. Thus, secondary structural constraints in the mRNA or changes in the exon sequence might prevent recognition of this alternative splice site to form B' mRNA in murine tissues.


Novel vectors for expression of cDNA encoding epitope-tagged proteins in mammalian cells.

Two composite constructs, pNHA and pCHA, are described. These plasmids are designed for the expression of cDNA in mammalian cells to produce proteins which are tagged with the hemagglutinin epitope sequence, YPYDVPDYA (HA1). The insertion of cDNA into the multiple cloning sites of these vectors has the advantage of 'tagging' the produced protein at either the N terminus or C terminus. To demonstrate the utility of these vectors, pNHA, containing a full-length cDNA (pNHRP33), was expressed in HeLa cells to produce a HA1-tagged peptide. The resulting peptide was purified from the whole-cell extracts by immunoprecipitation with an antibody to the tag (mAb12CA5). The mRNA was transcribed from a T7 promoter of the pNHRP33 construct, translated in a rabbit reticulocyte assay, and the protein product was purified using mAb12CA5 for the HA1 epitope. Among other possibilities, these vectors can be used to: (1) study protein-protein interactions in a mammalian transcription unit, (2) co-purify associated transcription factors, and (3) purify produced proteins when antibodies are not available.


[Sympathetic reflex dystrophy. Effectiveness of physical therapy treatment of Sudeck's syndrome].

To investigate the impairment of patients with reflex sympathetic dystrophy syndrome (RSDS) and to establish the effectiveness of two physiotherapeutic regimens in the treatment of this entity, 54 RSDS patients were examined clinically, radiologically and scintigraphically an average of 112 days after the triggering event. The patients were assigned to either of two treatment groups in accordance with the results of preliminary scintigraphic examinations. After physiotherapy comprising exercises and cryotherapy either with or without galvanic stimulation, a significant therapeutic effect on clinical and scintigraphic parameters was found in both treatment groups. Scanning, in combination with clinical diagnostic measures proved a valuable tool in the diagnostic evaluation, selection of treatment and follow-up in patients with RSDS.


Deletion delta F508 and haplotype analysis of CFTR gene region in Slovak CF patients.

Analysis of a sample of 50 unrelated cystic fibrosis (CF) patients and 46 nuclear families from Slovakia (Czechoslovakia) by the polymerase chain reaction and Southern hybridization revealed that the proportion of the delta F508 mutation was 58% in this population, and that the frequency of the B (i.e., KM19/XV2c [1-2]) haplotype was increased in both delta F508 and non-delta F508 CF chromosomes (98% and 46%, respectively). These results support the view that the trans-European gradient of the delta F508 frequency is of a geographical rather than of an ethnic origin, and that in Slavonic populations, there exists an as yet unidentified but frequent CF mutation other than delta F508, associated with the B haplotype.


A novel sickle cell mutation of yet another origin in Africa: the Cameroon type.

The sickle cell mutation (beta s) arose as at least three independent events in Africa and once in Asia, being termed the Senegal, Benin, Bantu and Indian types respectively. An investigation in Cameroon was carried out to determine whether the atypical sickle genes observed in the neighboring countries are the result of recombination or the presence of a sickle cell mutation of a different genetic origin. It was conducted on 40 homozygous SS patients followed at the Blood Transfusion Center in the capital city of Yaoundé. On 80 beta s chromosomes, 13 exhibited a novel polymorphic pattern that was observed three times in the homozygous state. This chromosome contains an A gamma T gene. The restriction fragment length polymorphism haplotype is different from all the other beta s chromosomes in both the 5' and 3' regions, but has previously been reported in sporadic cases. The (AT)8(T)5 sequence in the -500 region of the beta gene is specific and different from that of the Senegal, Benin, Bantu or Indian beta s genes. All the carriers of this specific chromosome belong to the Eton ethnic group and originate from the Sanaga river valley. This observation strongly argues for yet another independent origin of the sickle cell mutation in Africa, here referred to as the "Cameroon type". The Benin haplotype and a Benin/Bantu recombinant haplotype have been observed in the other studied populations: Ewondo, Bamiléké, Bassa, Yambassa and Boulou.


Immunorecognition of different ganglioside epitopes on human normal and melanoma tissues.

There is increasing evidence that cell-surface gangliosides play a role in tumor growth, progression and metastases. In order to determine the frequency of ganglioside GD3 in patients with metastatic malignant melanoma for further therapeutic trials, GD3 ganglioside expression was determined in 119 tissue samples. Of these melanomas, 93% (111/119) were R-24-positive, which indicates the value of this diagnostic marker for melanoma. To study the structural epitopes of gangliosides, 10 ganglioside antibodies with defined specificities and affinities were tested on over 100 fresh-frozen tissue specimens of human normal and melanoma tissues. All the antibodies tested recognize the ganglioside GD3, but vary in their cross-reactivity with other gangliosides. According to their epitope specificity, they can be divided into 5 groups. For example, the antibodies Z-21 and A-4 react like the previously established MAb R-24 with gangliosides GD3 and GQlb, and one MAb (Q-4) detects all gangliosides containing 2 connected sialic acids (GD3, GD2, GDlb, GTlb, GQlb). Specificity on TLC does not always correlate with specificity to melanoma tissues and vice-versa. For example, MAb A-4, which recognizes only GD3 and GQlb on TLC, shows no specific reactivity on tissues. Furthermore, antibodies with the same ganglioside specificity do not have the same staining pattern on human tissues. For example, MAb Z-21, which is directed against the same gangliosides as MAb R-24 on TLC, does not cross-react with as many neuroectodermal tissues as MAb R-24. Because of their distinct properties, some of these antibodies may be even more useful for immunodiagnosis and immunotherapy of malignant melanoma than MAb R-24.


Studies of clonal cell lines developed from primary breast cancers indicate that the ability to undergo morphogenesis in vitro is lost early in malignancy.

In an attempt to obtain cell lines from the different components found in primary breast cancers, we used a low-calcium medium to culture epithelial cells of mixed phenotype and a recombinant T antigen containing retrovirus to immortalize cells found in these cultures. In each case, the histology of the sample used for culture was examined in detail and the best growth was obtained from samples associated with a substantial in situ or benign component and from lobular rather than ductal carcinomas. Clonal cell lines were developed from each of 4 tumours: 1 infiltrating ductal (tumour number 2), 2 infiltrating lobular (tumours 3 and 5) and 1 mucoid (tumour 6). To try to identify the phenotype and origin of the cell lines, immunohistochemical markers, histological analysis of tissue sections and behavioural markers were used. All the cell lines expressed mainly luminal epithelial cell markers, but the basal epithelial keratin, keratin 14, was also expressed homogeneously or heterogeneously. Growth in agar was seen with some but not all cell lines derived from only 1 tumour (tumour 5) and tumour development in nude mice was observed (with low efficiency) with cell lines from only 1 tumour (tumour 6). The data suggest that the cell lines obtained from the infiltrating ductal carcinoma (tumour 2) developed from cells cultured from the associated benign component, while the cell lines from tumours 3, 5 and 6 may each have developed from a cell in an early stage of malignancy. When tested for their ability to undergo morphogenesis on extracellular matrix components, cell lines from tumour 2 made well-developed ductal-alveolar-like structures, while those from the other tumours did not.


Dipyridamole enhances an anti-proliferative effect of interferon in various types of human tumor cells.

The anti-proliferative activity of human interferon (HuIFN) was enhanced by dipyridamole, 2,6-bis-(diethanolamino)-4,8-dipiperidinopyrimido-[5,4-d]-py rimidine, when tested against various human tumor cell lines, including KT (breast carcinoma), PLC/PRF/5 (hepatoma), MGC-I, U251-SP and T98 (glioma), HAC-2 and SHIN-3 (ovarian carcinoma), and MM-ICB (melanoma). The enhancement occurred irrespective of the kind of HuIFN used (alpha, beta or gamma) and the original degree of susceptibility of the cells to HuIFN. Even low doses down to 0.01 microM of dipyridamole that had no intrinsic anti-proliferative activity could enhance the effect of HuIFN. The enhancement of HuIFN effects seems not to be caused by induction of HuIFN production, because neither anti-viral activity nor HuIFN antigens were detected in culture medium in cells treated with dipyridamole. Mopidamole, a derivative of dipyridamole lacking one piperidine residue, produced little enhancement of the effects of HuIFN. Among ovarian cancer cell lines tested, the enhancement of the activity of HuIFN by dipyridamole for HAC-2 and SHIN-3 cells was equivalent to or greater than that for 3 chemotherapy agents (adriamycin, vincristine, and a camptothecin derivative). However, neither HOC-21 ovarian cancer cells nor HEC-1 endometrial adenocarcinoma cells were susceptible to any combinations. When MGC-1, U251-SP, and HAC-2 cells were injected into nude mice, the growth of tumors was more markedly inhibited by the subcutaneous administration of HuIFN in combination with oral administration of dipyridamole than by the HuIFN alone. Thus, this combination therapy seems to be worth trying for human cancer, although the enhancement of the effects of HuIFN by dipyridamole varied among the cell lines examined.


Inhibition of tumor-induced angiogenesis by the administration of recombinant interferon-gamma followed by a synthetic lipid-A subunit analogue (GLA-60).

The effect of the administration of recombinant interferon-gamma (rIFN-gamma) and a synthetic lipid A subunit analog (GLA-60) on angiogenesis induced by B16-BL6 melanoma was examined in syngeneic C57BL/6 mice. Intravenous administration of rIFN-gamma followed by GLA-60 (referred to as rIFN-gamma/GLA-60) induced endogenous production of tumor necrosis factor (TNF). This treatment on day 3 after tumor inoculation caused a marked decrease in the number of vessels oriented towards the tumor mass (angiogenic response) and tumor size over a period of 9 days. In contrast, neither rIFN-gamma nor GLA-60 alone, nor GLA-60/rIFN-gamma (reverse sequence of administration), which is unable to induce the production of TNF in the serum, had any effect. Sera induced by the treatment with rIFN-gamma/GLA-60, and recombinant TNF, inhibited the in vitro growth of lung endothelial cells which is considered to be one of the essential events in tumor neovascularization. Multiple i.v. treatments with rIFN-gamma/GLA-60 on days 5, 8 and 11 after s.c. implantation of tumor significantly inhibited primary tumor growth by the amputation time (day 20) and lung metastasis of B16-BL6 cells on day 34, while other treatment modalities had no such effect. Our results indicate that inhibition of lung-tumor metastasis by rIFN-gamma/GLA-60 treatment may be partly due to the inhibition of tumor-associated angiogenesis.


The effect of retinoic acid on chemosensitivity of PA-1 human teratocarcinoma cells and its modulation by an activated N-ras oncogene.

Combination of chemotherapeutic drugs with agents that induce cell differentiation is a possible means of improving cancer chemotherapy. To explore this approach we used 4 cell lines established from the human teratocarcinoma-derived cell line PA-1; 2 retinoic acid (RA)-sensitive lines compared to 2 RA-resistant lines transformed by an activated N-ras oncogene. Equal numbers of colony-forming cells were exposed for 72 hr to 10(-6)M RA and subsequently to a range of concentrations of cisplatinum, etoposide or bleomycin. Enhanced cytotoxicity of cisplatin and etoposide (3- to 5-fold) was observed in the N-ras-transformed cell lines compared to the non-transformed lines. Treatment with RA caused an increase in the cytotoxicity of all 3 drugs to the 2 RA-sensitive cell lines. In contrast, a reduction of cytotoxicity was observed in the 2 N-ras-transformed lines. Our results indicate that sensitivity to cytotoxic agents can be increased by RA in RA-sensitive cells, but the opposite effect is seen in N-ras transformed, RA-resistant cells. Therefore, a general rationale for combination therapy with RA and cytotoxic drugs cannot be inferred.


Polymeric microballoons as ultrasound contrast agents. Physical and ultrasonic properties compared with sonicated albumin.

Air-filled polymeric microballoons were prepared with number-mean diameters of approximately 3 microns, volume-mean diameters of approximately 5 microns, and narrow particle-size distributions (standard deviation [SD] = 1.2 microns in number and SD = 2.0 microns in volume). More than 99% of the particles were below 8 microns. These particles were found to be highly echogenic for ultrasound, showing backscatter coefficients at 7.5 MHz, similar to the ones obtained with sonicated albumin microspheres. However, at 2.25 MHz, microballoons were less echogenic than albumin microspheres. These results are consistent with ultrasound attenuation measurements, which showed a maximum at 8 to 9 MHz for the microballoons compared with a reported value of 3.5 to 4.5 MHz for albumin microbubbles. Polymeric microballoons were found to be stable in plasma or under applied pressure as evidenced by unchanged particle concentration and echogenicity. Albumin microspheres were particularly unstable to applied pressure (150 mm Hg) and showed a rapid decrease in both particle counts and echogenicity.


Epithelial modulation of allergen and drug effects in guinea pig airways.

The effect of egg albumin (EA) challenge on tracheal tube preparations from sensitized guinea pigs was studied with regard to EA permeability, histamine release and penetrability, and the contractile response of the preparation. We used a plethysmographic method that allowed simultaneous measurement of changes in smooth muscle tension and collection of samples for determination of mediators. Our results clearly show that epithelial damage potentiates the contractile response to histamine, potassium ions, and acetylcholine. Epithelial damage did not alter the maximal contractile response in preparations challenged with high antigen concentrations (EA, 1 mg/ml), but histamine release measured in the perfusate increased substantially. The permeability of the preparations to EA was greater when the epithelium was damaged. No increase in the permeability in response to the EA challenge was observed. The present study has demonstrated that guinea pig airway epithelium constitutes a barrier for both antigen and drugs. We also present a method for recording contractile responses from intact whole tracheal preparations, in which the epithelium can still act as a barrier, as is the case in vivo.


Induction of atrial natriuretic factor and myosin light chain-2 gene expression in cultured ventricular myocytes by electrical stimulation of contraction.

While hormonal stimuli and mechanical stretch can induced cardiac-specific gene expression and in some cases cellular hypertrophy, the relationship between myocyte contraction frequency, gene expression, and myocyte growth has not been well characterized. In this study a new model system was developed in which cultures of neonatal rat ventricular myocytes were subjected to long term pacing of contractions with pulsatile electrical stimulation. Myocytes submitted to electrical stimulation for 3 days displayed dramatic increases in cellular size and myofibrillar organization, and a 5-10-fold increase in the expression of the cardiac genes atrial natriuretic factor and myosin light chain-2. Atrial natriuretic factor expression induced by electrical stimulation of contractions was inhibited by nifedipine or W7, indicating a dependence on calcium influx and calmodulin activity. Phosphoinositide hydrolysis and cAMP formation were not affected by electrical stimulation suggesting that gene induction occurred independently of the activation of protein kinases C or A above basal levels. These findings show that the cellular events associated with contraction, such as changes in cytoplasmic free calcium levels and/or cellular stretch, may serve as important determinants of myocyte growth and cardiac gene expression.


A peptide corresponding to GPIIb alpha 300-312, a presumptive fibrinogen gamma-chain binding site on the platelet integrin GPIIb/IIIa, inhibits the adhesion of platelets to at least four adhesive ligands.

The platelet fibrinogen (Fg) receptor (GPIIb/IIIa) is an integrin which plays a critical role in hemostasis by recognizing at least the four adhesive ligands: Fg, fibronectin (Fn), vitronectin (Vn), and von Willebrand factor (vWf). We reported that residues 309-312 of GPIIb alpha appear to comprise at least part of a Fg binding site on the Fg receptor (Gartner, T. K., and Taylor, D. B. (1990) Thromb. Res. 60, 291-309). Here we report that the peptide GPIIb alpha 300-312 (G13) inhibits platelet aggregation and binds Fg and Vn. Significantly, this peptide inhibits the adhesion of stimulated platelets to Fg, Fn, Vn, and vWf, but not the adhesion of resting platelets to Fn. Thus, GPIIb 300-312 may constitute a specific but common recognition site on GPIIb/IIIa for both LGGAKQAGDV- and RGD-containing ligands.


Kinetics of inhibition of endogenous human immunodeficiency virus type 1 reverse transcription by 2',3'-dideoxynucleoside 5'-triphosphate, tetrahydroimidazo-[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thion e, and 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine derivatives.

Recently, tetrahydroimidazo-[4,5,1-jk][1,4]-benzodiazepin-2(1H)-one and -thione (TIBO) and 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) compounds have been shown to be potent, selective, and specific inhibitors of human immunodeficiency virus type 1 (HIV-1) replication in vitro. They interact with the reverse transcriptase of HIV-1 in a way different from that of previously studied reverse transcriptase (RT) inhibitors. We established an endogenous RT assay, starting from intact HIV-1 virions. This assay mimics the reverse transcription process in the HIV-infected cell more closely than RT assays with artificial templates. We investigated the inhibition of endogenous HIV-1 reverse transcription by the TIBO derivative (+)-(S)-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo [4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione (R-82150) in comparison with the HEPT derivative 5-ethyl-1-ethoxymethyl-6-(phenylthio)uracil (E-EPU) and 2',3'-dideoxyguanosine 5'-triphosphate. The kinetics and characteristics of RT inhibition by TIBO in the endogenous RT assay were similar to those found previously for the exogenous RT assay (following addition of exogenous template/primer); thus, RT inhibition by TIBO was specific for HIV-1 and the extent of RT inhibition was dependent on which of the four substrates (dATP, dTTP, dGTP, and dCTP) was present in limited concentrations. Of the three enzymatic activities, RNA-dependent DNA polymerization was preferentially inhibited, and inhibition was not competitive with respect to the natural substrates. HIV-1 RT behaved as an allosteric enzyme, which means that positive cooperativity for binding of the substrate was observed. TIBO behaved as an allosteric inhibitor by causing a concentration-dependent decrease in this cooperativity.


Induction chemotherapy in head and neck cancer: results of a phase III trial.

Between December 1982 and October 1986, 131 patients with stage II-III-IV squamous cell carcinoma of the oropharynx or oral cavity were randomized to induction chemotherapy, consisting of bleomycin (10 mg/m2/day in continuous infusion from day 1 to day 5), methotrexate (120 mg/m2 on day 2) followed by folinic acid, 5-fluorouracil (5 FU) (600 mg/m2 on day 2), and cisplatin (120 mg/m2 on day 4) every 4 weeks for a total of three cycles followed by definitive locoregional treatment versus locoregional treatment alone. The modalities of definitive treatment (radiotherapy +/- surgery) were chosen prior to randomization. A total of 116 patients were evaluable. Of 55 patients in the chemotherapy arm, four (7%) had a complete response (CR) and 23 (42%) a partial response (PR) following the induction regimen. At the completion of locoregional treatment, 76% (42 of 55) of patients in the experimental group were in CR compared to 89% (54 of 61) in the control group. There was no difference in survival, cause-specific survival, and pattern of relapse between both groups. The median survival was 22 months in the chemotherapy group and 29 months in the control group. Responders to chemotherapy did not fare better than nonresponders. Chemotherapy-related toxicities were few and most of them related to cisplatin which was reduced to 100 mg/m2 for 35 patients. There were no treatment-related deaths and, in the experimental arm of the trial, no increased morbidity from locoregional treatment. This induction regimen does not offer any advantages over standard treatment.


Basal and FSH-stimulated steady state levels of SGP-2, alpha 2-macroglobulin, and testibumin in culture media of rat seminiferous tubules at defined stages of the epithelial cycle.

Production of several proteins by rat Sertoli cells is dependent on the stage of the cycle of the seminiferous epithelium. The authors have determined steady state levels and follicle-stimulating hormone responsiveness of three Sertoli cell products in culture media of rat seminiferous tubule segments at different stages of the epithelial cycle: SGP-2 (sulfated glycoprotein-2), alpha 2-macroglobulin, and testibumin. Basal SGP-2 levels were twofold higher in stages VII through VIII compared with stages XIII to I to VI (P less than 0.05). Highest basal alpha 2-macroglobulin levels were found in stages II through VIII; this was about 35% greater than in stages XIII through I of the cycle (P less than 0.05). Basal testibumin levels were twofold higher in stages II through VI compared with stages IX through XII of the cycle. Follicle-stimulating hormone had no effect on SGP-2, but by contrast it (50 mg/L) increased the level of alpha 2-macroglobulin significantly (P less than 0.05) in stages XIII through I. Follicle-stimulating hormone treatment (10 mg/L) elevated testibumin levels at each stage-pool by about 40% (P less than 0.05). The current results using staged tubular segments in vitro demonstrate cyclic basal steady-state levels of the three proteins along the seminiferous tubules and follicle-stimulating hormone regulation of alpha 2-macroglobulin and testibumin.


Desensitization of the human V2 vasopressin receptor. Homologous effects in the absence of heterologous desensitization.

Three main pathways have been implicated in desensitization of receptors that stimulate adenylylcyclase (AC): cAMP-mediated phosphorylation; cAMP-independent phosphorylation, and receptor internalization. Cell lines derived from the murine Ltk- cell were found useful in exploring the contribution of cAMP-dependent phosphorylation in V2 vasopressin receptor desensitization. The HTB-2 cell expresses the human V2 vasopressin receptor, introduced by transfection of human genomic DNA, and the prostaglandin E1 (PGE1) receptor, endogenous to the Ltk- cell. The A7 cell expresses the hamster beta 2-adrenoceptor, which undergoes the above-mentioned desensitization processes. Treatment of HTB-2 cells with arginine-vasopressin (AVP) had no effect on AC responsiveness to PGE1, but promoted desensitization of the AVP response. This was seen as a 5-6-fold right shift in the dose-response curves for AVP action (cAMP accumulation in intact cells and AC stimulation in homogenates and isolated membranes) and in a decrease in the maximum effect of AVP on these parameters. AVP treatment caused a decrease in cell surface receptors to approximately 75% of control without changes in KD, as determined by Scatchard analysis. When cAMP was increased by treatment with 10 microM PGE1 and isobutylmethylxanthine, desensitization of the PGE1 receptor was observed but not of the AVP receptor. In A7 cells the same treatment caused, as expected, a 3-fold right shift in the dose-response curve for AC stimulation by isoproterenol, indicating that L cells can mediate heterologous desensitization. These data demonstrate that the V2 vasopressin and the PGE1 receptors undergo homologous desensitization in the absence of cAMP-mediated phosphorylation and that this component is not required for vasopressin receptor internalization.


High affinity inhibition of Ca(2+)-dependent K+ channels by cytochrome P-450 inhibitors.

The Ca(2+)-dependent K+ channel of human red cells was inhibited with high affinity by several imidazole antimycotics which are potent inhibitors of cytochrome P-450. IC50 values were (in microM): clotrimazole, 0.05; tioconazole, 0.3; miconazole, 1.5; econazole, 1.8. Inhibition of the channel was also found with other drugs with known cytochrome P-450 inhibitory effect. However, no inhibition was obtained with carbon monoxide (CO). This suggests that, given the high selectivity of the above inhibitors for the heme moiety, a different but closely related to cytochrome P-450 kind of hemoprotein may be involved in the regulation of the red cell Ca(2+)-dependent K+ channel. Clotrimazole also inhibited two other charybdotoxin-sensitive Ca(2+)-dependent K+ channels, those of rat thymocytes (IC50 = 0.1-0.2 microM) and of Ehrlich ascites tumor cells (IC50 = 0.5 microM). Imidazole antimycotics inhibit also receptor-operated Ca2+ channels (Montero, M., Alvarez, J. and García-Sancho, J. (1991) Biochem. J. 277, 73-79). This suggests that both Ca2+ and Ca(2+)-dependent K+ channels might have a similar regulatory mechanism involving a cytochrome.


Kinetics of inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by the novel HIV-1-specific nucleoside analogue [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5 "- (4"-amino-1",2"-oxathiole-2",2"-dioxide)thymine (TSAO-T).

[2',5'-Bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro- 5"-(4"-amino-1",2"-oxathiole-2", 2"-dioxide)thymine (TSAO-T) is a representative of a novel class of nucleoside analogues that are endowed with a potent and specific activity against human immunodeficiency virus (HIV) type 1 and are targeted at the HIV-1 reverse transcriptase (RT). Inhibition of HIV-1 RT by TSAO-T was reversible and noncompetitive with respect to dGTP as the substrate and poly(C).oligo(dG) as the template/primer. In contrast with the nonnucleoside derivatives tetrahydroimidazo-[4,5,1-jk][1,4]- benzodiazepin-2(1H)-thione (TIBO) (R-82150), nevirapine (BI-RG-587) and the HEPT derivative I-HEPU-SdM, TSAO-T was not inhibitory to HIV-1 RT in the presence of other homopolymeric template/primers. It did not interfere with the DNA-dependent DNA polymerase function of HIV-1 RT, HIV-2 RT, herpes simplex virus type 1 DNA polymerase, or Taq polymerase. However, TSAO-T proved inhibitory to the HIV-1 RT reaction primed by Escherichia coli 16S/23S rRNA, irrespective of the nature of the radiolabeled 2'-deoxynucleotide 5'-triphosphate (dNTP) used. TSAO-T does not act as a DNA chain terminator. It interacts with HIV-1 RT at a nonsubstrate (dNTP)-binding site.


Identification of the forms of insulin-like growth factor-binding proteins produced by human fibroblasts and the mechanisms that regulate their secretion.

Human fibroblasts secrete insulin-like growth factor-binding proteins (IGFBPs) that can modify insulin-like growth factor (IGF) I action. We have determined the molecular identities of three forms of IGFBPs that are secreted by human fibroblasts in vitro. Ligand blot analysis of fibroblast conditioned media revealed that the M(r) 43,000 and 39,000 forms were the most abundant, but that M(r) 31,000 and 24,000 forms were also present. An antiserum that was specific for IGFBP-5 reacted with the M(r) 31,000 form, and an IGFBP-4-specific antiserum recognized only the M(r) 24,000 form. The M(r) 39,000 and 43,000 forms were detected by IGFBP-3 antiserum. Further proof that fibroblasts synthesized these forms of IGFBPs was obtained by Northern blotting. A cDNA probe for IGFBP-3 hybridized with a 2.4-kilobase (kb) transcript, whereas a cDNA probe for IGFBP-5 recognized a single 6.0-kb transcript, and an IGFBP-4 cDNA probe recognized 2.2- and 2.0-kb transcripts. IGF-I and -II caused a minimal (less than 43%) increase in IGFBP-5 mRNA abundance and had no effect on IGFBP-4 mRNA abundance. IGF-I and -II (100 ng/ml) stimulated 6-8-fold increases in IGFBP-5 levels, whereas IGFBP-4 was inhibited. Insulin failed to elicit any change in IGFBP-5, suggesting that binding of the IGFs to IGFBPs was required to detect the increase. Immunoblotting for IGFBP-5 revealed an M(r) 23,000 (non-IGF-I-binding) fragment. To determine if the IGFs were influencing proteolytic degradation of IGFBP-5, pure IGFBP-5 was added to fibroblast cultures and incubated for 4 h at 37 degrees C. The amount of fragment formation was attenuated by the presence of IGF-I and -II, but not insulin, suggesting that this is a mechanism by which the IGFs act to modulate IGFBP-5 concentration. In contrast to the IGFs, forskolin, which increased IGFBP-4 and -5 mRNA abundance and secretion, had no effect on fragment formation. The results show that human fibroblasts synthesize and secrete IGFBP-3, -4, and -5 and that changes in intracellular cAMP regulate synthesis, whereas the IGFs regulate IGFBP-4 and -5 levels by post-transcriptional mechanisms.


Mapping of binding sites for heparin, plasminogen activator inhibitor-1, and plasminogen to vitronectin's heparin-binding region reveals a novel vitronectin-dependent feedback mechanism for the control of plasmin formation.

Vitronectin (VN) has been implicated as a major matrix-associated regulator component of plasminogen activation by serving as a potent stabilizing cofactor of plasminogen activator inhibitor-1 (PAI-1). The direct binding of heparin, plasminogen as well as PAI-1 in its latent and active form to immobilized VN was studied in the absence or presence of competitors. Monoclonal antibodies against the carboxyl-terminal portion of VN inhibited both PAI-1 and plasminogen binding, whereas heparin, heparan sulfate with a high degree of sulfation, or dextran sulfate interfered with PAI-1 binding (KD = 20 nM) only. Utilizing synthetic peptides encompassing overlapping sequences of the heparin-binding domain of VN, adjacent heparin and PAI-1-binding sites were localized within the sequence 348-370 of VN. Although a number of other serine protease inhibitors which do not form binary complexes with VN contain a reactive-site Ser at their P1'-position, a reactive-site P1' mutant of PAI-1 (Met----Ser) showed comparable if not increased binding to VN. Binding of Lys-plasminogen and active-site-blocked plasmin was at least 10-fold higher in affinity (KD = 85-100 nM) compared to Glu-plasminogen (KD approximately 1 microM) and could be inhibited by lysine analogs but not by glycosaminoglycans or PAI-1, indicating that heteropolar plasmin(ogen) binding of VN occurs to an adjacent segment upstream to the heparin and PAI-1-binding sites. This contention was further supported in binding studies with plasmin-modified VN which lost both heparin and PAI-1 binding but exhibited 2-3-fold higher capacity to bind plasminogen. The essential plasmin(ogen)-binding site was mapped by ligand blot analysis to the carboxyl-terminal portion of proteolytically trimmed VN (M(r) = 61,000). Moreover, treatment of the extracellular matrix of human umbilical vein endothelial cells with plasmin resulted in partial degradation of matrix-associated VN and concomitant release of PAI-1, but increased the ability of the matrix by about 2-fold to bind plasminogen. These results are indicative of differential interactions of VN with components of the plasminogen activation system, whereby plasmin itself may provoke the switch of VN from an anti-fibrinolytic into a pro-fibrinolytic cofactor. This process reflects a novel role for the adhesive protein and its degradation product(s) in the possible feedback regulation of localized plasmin formation at extracellular sites.


Immunoglobulin-mediated phagocytosis by human monocytes requires protein kinase C activation. Evidence for protein kinase C translocation to phagosomes.

This study has investigated the role of protein kinase C (PKC) activation in IgG-mediated phagocytosis by human monocytes. Incubation of monocytes with IgG-opsonized targets increased membrane-associated PKC approximately 2-fold. Kinetic studies showed that the translocation of PKC to membrane occurred before significant ingestion took place. The pharmacologic PKC inhibitor H7 inhibited IgG-dependent ingestion with ID50 of 20 microM, while the structurally related isoquinoline sulfonamide HA1004 had no effect at this concentration. Staurosporine and calphostin C, PKC inhibitors which have different mechanisms of actions than H7, also inhibited ingestion. Depletion of PKC by prolonged incubation with phorbol esters also inhibited phagocytosis, and dose-response curves showed a strong correlation between the extent of PKC depletion and the extent of inhibition of ingestion. Finally, phagosomes were isolated by sucrose density centrifugation of cells disrupted 5 min after the initiation of phagocytosis. Measurement of PKC activity and immunoreactivity in the phagosomes showed that PKC was concentrated in the phagosome membrane approximately 5-fold compared to the uninvolved plasma membrane. Together, these data suggest that PKC activation is an early, essential step in the efficient ingestion of IgG-opsonized targets by monocytes.


The alpha-operon equivalent genome region in the extreme halophilic archaebacterium Haloarcula (Halobacterium) marismortui.

The genome region of the extreme halophilic archaebacterium Haloarcula marismortui equivalent to the alpha-operon of Escherichia coli has been characterized. In H. marismortui, the alpha-operon was found to be located immediately upstream from the S9 gene cluster. The gene order in the halobacterial alpha-operon, given according to the gene products, is tRNA(Ser), HmaS13, HmaS4, HmaS11, and HmaRp alpha. Compared to the corresponding operon from E. coli, the halobacterial gene organization differs in (i) the presence of a gene for tRNA(Ser) (GCU), (ii) the reversed order of the genes for the ribosomal proteins HmaS11 and HmaS4, and (iii) the absence of the gene coding for the ribosomal protein L17. The primary structure of HmaRp alpha shows high similarity to a subunit of eukaryotic RNA polymerase II (YeaRpB3, HsaRpB33), whereas the similarity to the eubacterial alpha-subunit of RNA polymerase is only weak.


Gene and pseudogene of the mouse cation-dependent mannose 6-phosphate receptor. Genomic organization, expression, and chromosomal localization.

The cation-dependent mannose 6-phosphate receptor (CD-MPR) is one of the two transmembrane proteins involved in transport of lysosomal enzymes. We have cloned the mouse CD-MPR gene and also a very unusual processed-type CD-MPR pseudogene. They are both present at one copy per haploid genome and map to chromosomes 6 and 3, respectively. Comparison of the complete 10-kilobase (kb) sequence of the functional gene with the cDNA indicates that it contains seven exons. Exon 1 encodes the 5'-untranslated region of the mRNA, the others (exons 2-7) encode the luminal, transmembrane, and cytoplasmic domains of the CD-MPR. Exon 7 also contains a 1.2-kb-long 3'-untranslated region of the mRNA. A unique transcription-initiation site was determined by primer extension of mouse liver mRNA. The promoter elements in the 5' upstream region of this site resemble those contained in genes constitutively transcribed. However, Northern blot analysis demonstrates that the CD-MPR is variably expressed in adult mouse tissues and during mouse development. The pseudogene, which is flanked by direct repeats, is almost colinear with the cDNA indicating that it presumably arose by reverse transcription of an mRNA. However, the pseudogene differs from the cDNA. It contains at its 5' end, an additional 340-nucleotide (nt) sequence homologous to the promoter region of the functional gene. This sequence exhibits some promoter activity in vitro. Furthermore, a 24-nt insertion interrupts the region homologous to the 5'-noncoding region of the cDNA. In the functional gene, this 24-nt sequence occurs between exon 1 and 2, where it is flanked by typical consensus sequences of exon/intron boundaries. Therefore, it may represent an additional exon of the functional gene. These two features of the pseudogene suggest that expression of the CD-MPR gene may be regulated by use of different promoters and/or alternative splicing.


Replacement of negative by positive charges in the presumed membrane-inserted part of diphtheria toxin B fragment. Effect on membrane translocation and on formation of cation channels.

Diphtheria toxin B fragment is capable of forming cation-selective channels in the plasma membrane. Such channels may be involved in the translocation of the toxin A fragment to the cytosol. Seven negatively charged amino acids in the B fragment were replaced one by one by lysines, followed by studies of cytotoxicity and channel-forming ability of the different mutants. The mutant D392K showed a strong reduction in binding to cell surface receptors. Of the six mutants that showed wild-type binding affinity, the two mutants D295K and D318K were very inefficient in forming channels. These two mutants had the lowest ability to mediate A fragment translocation. The mutant E362K was able both to induce cation channel formation and to mediate A fragment translocation at a higher pH value than the wild-type B fragment. The results support the notion that formation of cation channels is of importance for the translocation of the A fragment across the plasma membrane, and they indicate that the pH requirement for translocation of the A fragment to the cytosol is partly determined by the B fragment.


Analysis of the importance of arginine 102 in neutral endopeptidase (enkephalinase) catalysis.

Neutral endopeptidase 24.11 contains an active site arginine believed to function in substrate binding. This arginine is thought to form an ionic interaction with the COOH-terminal carboxylate of NEP substrates. The functionality of arginine 102 has been investigated by using site-directed mutagenesis to produce mutants in which this residue was converted to a lysine, glycine, glutamine, or glutamate. All of the mutants exhibited essentially full activity as determined with a synthetic peptide amide, glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide. In contrast, activity was detected only with the wild-type enzyme and the lysine mutant using a synthetic substrate containing a free COOH-terminal carboxylate, dansyl-Gly-Trp-Gly. Inhibition studies with the physiologically active peptide substrates substance P, endothelin, and angiotensin I, as well as substance P free acid, [D-Ala2,Leu5]enkephalin, and [D-Ala2,Leu5]enkephalinamide indicated a lack of importance of arginine 102 in substrate binding. With [D-Ala2,Met5]enkephalin and the chemotactic peptide, N-formyl-Met-Leu-Phe, a significant decrease in affinity is observed with the arginine 102 mutants. These results suggest that the contribution of arginine 102 to substrate binding is dependent upon the strength of other subsite interactions. Examination of dipeptides as inhibitors indicates that the nature and orientation of the P'2 residue is important in determining the strength of the interaction of arginine 102 with its substrates.


Influence of subunit-specific antibodies on the activity of the F0 complex of the ATP synthase of Escherichia coli. I. Effects of subunit b-specific polyclonal antibodies.

Incubation of F1-stripped everted membrane vesicles with antibodies against subunit b of the ATP synthase from Escherichia coli resulted in an inhibition of the binding of F1 to F0, whereas the proton translocation remained unaffected. Incubation of unstripped everted membrane vesicles with anti-b antibodies resulted in a partial loss of F1, and the remaining membrane-bound ATP-hydrolyzing activity is uncoupled from proton translocation. Similar results were obtained when F(ab')2 or Fab fragments were used. The immunoblot analysis of truncated b' subunits different in length showed that the antigenic determinants are located in the carboxyl-terminal half of the polypeptide chain.


Influence of subunit-specific antibodies on the activity of the F0 complex of the ATP synthase of Escherichia coli. II. Effects of subunit c-specific polyclonal antibodies.

After incubation of F1-stripped everted membrane vesicles with antibodies against subunit c of the ATP synthase of Escherichia coli the proton translocation through the open F0 channel was blocked. Rebinding of F1 to those vesicles is affected by the antibody concentration used. In general, the use of F(ab')2 or Fab fragments prepared from anti-c antibodies gave similar results. However, using Fab fragments a higher amount of antigenic binding sites was necessary to block the F0 complex completely, whereas extremely low amounts of Fab fragments were necessary to inhibit the binding of F1. This can be explained by an antigenic determinant of subunit c, which is only accessible to the smaller Fab fragments with a molecular mass of approximately 50,000. Incubation of F1-containing everted membranes with anti-c antibodies showed that the binding of the antibodies resulted in a displacement of F1, while simultaneously the proton translocation through F0 has been blocked. Such a displacement can only be observed after incubation with IgG molecules or F(ab')2 fragments. Fab fragments were not able to displace the F1 part, indicating that the ability of antibodies and F(ab')2 fragments to produce cross-links is responsible for the loss of F1 from the membranes.


Identification of a factor in fetal bovine serum that stabilizes the cumulus extracellular matrix. A role for a member of the inter-alpha-trypsin inhibitor family.

Fetal bovine serum (FBS) has been widely used in the culture of cumulus-oocyte complexes, since it is believed that FBS stabilizes the expanding cumulus extracellular matrix. In this study, we have identified a factor in FBS that is responsible for this effect. FBS was first fractionated by stepwise precipitation with ammonium sulfate followed by high performance liquid chromatography (HPLC) on a gel filtration column. Active fractions, as determined by their ability to stabilize the cumulus extracellular matrix in a bioassay system, were further purified by HPLC on DEAE ion-exchange and gel filtration columns. The purified factor was exhibited as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr 150,000 and a single peak on a C-8 reverse-phase HPLC. The sequence of the first 14 NH2-terminal amino acids was determined by Edman degradation, revealing significant sequence identity of the bovine serum factor with the light chain shared by members of the inter-alpha-trypsin inhibitor family. Western blot analysis using anti-human I alpha I IgG shows that the antibody reacts positively with the purified factor, and immunodepletion of FBS with anti-I alpha I antibody agarose eliminated its ability to stabilize the extracellular matrix. This factor is found in mouse follicular fluid collected 6 h following human chorionic gonadotropin injection to stimulate ovulation, but not in unstimulated mice. Anti-I alpha I-positive epitopes were also localized within the cumulus extracellular matrix of mouse preovulatory follicles in immunocytochemical preparations using anti-human I alpha I IgG, supporting the possibility that this molecule or molecules may diffuse into follicular fluid after an ovulatory stimulus to act as structural linkers that ensure normal cumulus expansion, through stabilization of the cumulus extracellular matrix thus supporting the process of ovulation.


Albumin-binding surfaces for implantable devices.

Surfaces of implantable and blood contact-devices accumulate adsorbed and denatured proteins. This anomalous layer of proteins may help trigger unwanted events such as activation of coagulation systems and, perhaps, chronic inflammation. Because, in many experimental systems, the purposeful coating of surfaces with albumin will biologically "passivate" materials, we have attempted to develop polymers which, when exposed to blood or body fluids, will spontaneously, selectively, and reversibly adsorb host albumin. We report here a novel derivatization technique for increasing the albumin affinity of implantable polyetherurethane (PU). The technique is based on the incorporation of high-molecular-weight dextran to which the albumin-binding dye Cibacron Blue is covalently attached. Somewhat surprisingly, the amounts of human albumin adsorbed by Blue Dextran-modified and unmodified PU are quite similar. There are, however, important differences. First, the binding of albumin to derivatized PU is specific and not readily blocked by proteins in albumin-depleted human serum. Second, the majority of albumin associated with derivatized PU appears to be reversibly bound. Third, the binding of albumin to derivatized PU evidently is mediated primarily through ligand-specific binding of the protein to the albumin-binding dextran-dye conjugate. We conclude that it is possible to produce implantable polymers having surfaces which display albumin-binding dyes that selectively and reversibly bind albumin. Materials with this property, when implanted or exposed to blood, should form an infinitely renewable coating of albumin derived from physiologic fluids. This surface modification strategy may spawn a new generation of implantable materials with improved biologic compatibility.


Situational variation in problem behavior at home and school in attention deficit disorder with hyperactivity: a factor analytic study.

The present investigation assesses situational variation in problem behaviors of children with Attention Deficit Disorder with Hyperactivity. Principal-component factors (VARIMAX rotation) were computed for the Home/School Situations Questionnaires (HSQ/SSQ). For the HSQ, four factors emerged, accounting for 61% of variance. For the SSQ, three factors emerged, accounting for 68% of variance. Factors were similar to those previously derived with non-referred children. To assess discriminative power, factors were compared (t-tests) to previous data. All factors significantly discriminated ADD/H from non-referred children. These data suggest the factors add to the utility of the HSQ/SSQ in assessing ADD/H.


Listening preferences in regard to speech in four children with developmental disabilities.

In a previous experimental study of children's listening responses in regard to speech sounds, autistic children showed preferential patterns which were in sharp contrast to a group of mentally-handicapped and normally-developing children. The present study reports the data obtained for four children whose clinical diagnosis (1) became available only some time after the study was carried out, and/or (2) differed from previously recorded clinical impressions. The results obtained support previous findings according to which a lack of attraction to speech sounds appears to be a feature of young autistic children's overall disregard to people. The present technique is discussed as a viable method to examine experimentally young autistic children's social unresponsiveness.


Induction of active and adoptive relapsing experimental autoimmune encephalomyelitis (EAE) using an encephalitogenic epitope of proteolipid protein.

Proteolipid protein (PLP) is a major component of the central nervous system (CNS) myelin membrane and has been shown to induce acute experimental autoimmune encephalomyelitis (EAE) in genetically susceptible animals. Here we describe conditions by which a relapsing-remitting form of EAE can be reliably induced in SJL/J mice either actively immunized with the major encephalitogenic PLP peptide, PLP13-151(S), or following adoptive transfer of PLP139-151(S)-specific T cells. The disease follows a reliable relapsing-remitting course with acute clinical signs first appearing 6-20 days after priming or transfer and relapses first appearing at 30-45 days. The initial onset of disease correlates with delayed-type hypersensitivity (DTH) reactivity specific for PLP139-151(S), in the apparent absence of T cell reactivity to the major myelin basic protein (MBP) peptide. Histologically, both the active and adoptive forms of the disease are characterized by extensive mononuclear cell infiltration and severe demyelination of the CNS. These results suggest that T cell responses specific for PLP139-151(S) are sufficient to induce clinical and histological R-EAE in SJL/J mice. This model should prove useful for examination of the cellular and molecular events involved in clinical relapses and perhaps in determining the role of PLP-specific T cell responses in multiple sclerosis (MS).


Human piebald trait resulting from a dominant negative mutant allele of the c-kit membrane receptor gene.

Human piebald trait is an autosomal dominant defect in melanocyte development characterized by patches of hypopigmented skin and hair. Although the molecular basis of piebaldism has been unclear, a phenotypically similar "dominant spotting" of mice is caused by mutations in the murine c-kit protooncogene. In this regard, one piebald case with a point mutation and another with a deletion of c-kit have been reported, although a polymorphism or the involvement of a closely linked gene could not be excluded. To confirm the hypothesis that piebaldism results from mutations in the human gene, c-kit exons were amplified by polymerase chain reaction from the DNA of 10 affected subjects and screened for nucleotide changes by single-stranded conformation polymorphism analysis. In one subject with a variant single-stranded conformation polymorphism pattern for the first exon encoding the kinase domain, DNA sequencing demonstrated a missense mutation (Glu583----Lys). This mutation is identical to the mouse W37 mutation which abolishes autophosphorylation of the protein product and causes more extensive depigmentation than "null" mutations. In accord with this "dominant negative" effect, the identical mutation in this human kindred is associated with unusually extensive depigmentation. Thus, the finding of a piebald subject with a mutation that impairs receptor activity strongly implicates the c-kit gene in the molecular pathogenesis of this human developmental defect.


Distinction of human immunodeficiency virus type 1 neutralization and infection enhancement by human monoclonal antibodies to glycoprotein 120.

There is increasing evidence that sera from HIV-1-infected individuals contain antibodies that enhance infection by HIV-1 in vitro. Previous work has demonstrated that complement receptors on T lymphoid cells and Fc receptors for IgG (Fc gamma R) on monocytic cells are required for enhanced infection by antibody-complexed HIV-1. Characterization of such infection-enhancing antibodies is essential because immunogenic epitopes which induce enhancing antibodies should be excluded from HIV-1 vaccines. This study was conducted to identify enhancing antibodies involved in Fc R-mediated enhancement of HIV-1 infection employing IgG human monoclonal antibodies (HMAbs) reactive against gp120 of HIV-1, which were produced by B cell lines derived from an HIV-1-infected individual. A potent neutralizing HMAb N70-1.5e did not enhance infection by HIV-1 (IIIB and MN strains), whereas HMAb N70-2.3a mediated enhancement of HIV-1 infection, but had little neutralizing activity. A competition radio immunoassay demonstrated that the two antibodies bind to distinct epitopes. These results indicated that enhancing and neutralizing antibodies can be induced by different epitopes on gp120, suggesting the potential for development of safe vaccines against HIV-1 by exclusion of immunogenic epitopes for enhancing antibodies. We made attempts to identify the epitope on gp120 that is recognized by the enhancing antibody N70-2.3a by using recombinant HIV-1 proteins and found that the antibody binds to a conformational site of nonvariable sequences in the carboxyl half (aa 272-509) of gp120.


Unique region of the minor capsid protein of human parvovirus B19 is exposed on the virion surface.

Capsids of the B19 parvovirus are composed of major (VP2; 58 kD) and minor (VP1; 83 kD) structural proteins. These proteins are identical except for a unique 226 amino acid region at the amino terminus of VP1. Previous immunization studies with recombinant empty capsids have demonstrated that the presence of VP1 was required to elicit virus-neutralizing antibody activity. However, to date, neutralizing epitopes have been identified only on VP2. Crystallographic studies of a related parvovirus (canine parvovirus) suggested the unique amino-terminal portion of VP1 assumed an internal position within the viral capsid. To determine the position of VP1 in both empty capsids and virions, we expressed a fusion protein containing the unique region of VP1. Antisera raised to this protein recognized recombinant empty capsids containing VP1 and VP2, but not those containing VP2 alone, in an enzyme-linked immunosorbent assay. The antisera immunoprecipitated both recombinant empty capsids and human plasma-derived virions, and agglutinated the latter as shown by immune electron microscopy. The sera contained potent neutralizing activity for virus infectivity in vitro. These data indicate that a portion of the amino terminus of VP1 is located on the virion surface, and that this region contains intrinsic neutralizing determinants. The external location of the VP1-specific tail may provide a site for engineered heterologous epitope presentation in novel recombinant vaccines.


Involvement of integrin alpha V gene expression in human melanoma tumorigenicity.

Human melanoma originates in the skin and can lead to wide-spread metastatic disease. Analysis of melanoma biopsy material has shown that the vitronectin receptor, integrin alpha v beta 3, is a specific marker of the most malignant cells, i.e., vertically invasive primary lesions or distant metastases (Albelda, S. M., S. A. Mette, D. E. Elder, R. Stewart, L. Damjanovich, M. Herlyn, and C. A. Buck. 1990. Cancer Res. 50:6757-6764), suggesting a role for this adhesion receptor in the malignant growth of human melanoma tumors. A cell model was established to analyze the role of alpha v integrins on the tumorigenicity of human melanoma. From M21 human melanoma cells, stable variants were selected that lack alpha v gene expression and thus fail to express integrin alpha v beta 3 (M21-L cells). These cells not only lost the ability to attach to vitronectin but showed a dramatic reduction in tumorigenicity when transplanted into athymic nude mice, compared with M21 cells, even though both cell types showed identical beta 1 integrin expression and growth properties in vitro. M21-L cells were stably transfected with a cDNA-encoding alpha v. This resulted in the functional expression of integrin alpha v beta 3 on these cells and completely restored their tumorigenicity. Thus, integrin alpha v gene expression and the resulting adhesive phenotype are directly involved in the proliferation of human melanoma in vivo.


The ability of gingival crevicular fluid acute phase proteins to distinguish healthy, gingivitis and periodontitis sites.

3 acute phase proteins, from the local gingival inflammatory response, were examined for their ability to distinguish healthy, gingivitis and periodontitis sites. Indirect competitive immunoassays were developed for the quantification of alpha 2-macroglobulin (alpha 2-M) and transferrin (TF), and for alpha 1-antitrypsin (alpha 1-AT), a double antibody sandwich assay was produced. Healthy (25), gingivitis (31) and periodontitis (28) sites were sampled with filter paper strips (2 x 13 mm) and the volume assessed with the Periotron 6000. The samples were eluted in phosphate-buffered saline and analyzed for alpha 2-M, alpha 1-AT and TF. The results were expressed as absolute amounts per sample (ng/30 s) and on a concentration basis (ng/microliter of GCF). Higher GCF absolute amounts of alpha 2-M, alpha 1-AT and TF were consistently obtained from diseased (gingivitis and periodontitis) sites than healthy sites (p less than 0.005). Absolute amounts of GCF alpha 2-M, alpha 1-AT and TF were increased in periodontitis sites over gingivitis sites, although these differences were not statistically significant (p greater than 0.1). When the results were expressed on a concentration basis, alpha 2-M levels from diseased sites were significantly higher than healthy sites (p less than 0.01). In addition, GCF TF concentration was increased in periodontitis compared to healthy sites (p = 0.03).


Parvalbumin-containing nonpyramidal neurons in intracortical transplants of rat hippocampal and neocortical tissue: a light and electron microscopic immunocytochemical study.

Previous immunocytochemical studies have shown that GABAergic nonpyramidal neurons of the rat hippocampus survive in intracerebral transplants. However, information is still lacking about the dendritic organization and the input synapses of these cells as well as their capacity to express the calcium-binding protein parvalbumin (PARV) under transplant conditions. In the present study, a monoclonal antibody against PARV was used to examine the dendritic morphology and the synaptic organization of parvalbumin-containing GABAergic neurons in hippocampal and dentate transplants. In addition, parvalbumin-containing nonpyramidal neurons were studied in neocortical transplants to compare the differentiation of grafted allocortical and neocortical nonpyramidal neurons. Tissue blocks of hippocampus and fascia dentata and of the parietal neocortex were taken from late embryonic rats (E 21 and E 16, respectively) and were transplanted into a cavity in the somatosensory cortex of young adult rats. After 3.5 or 7 months survival, the recipient brains were fixed by perfusion and immunostained for PARV. As in the hippocampal formation in situ, PARV-containing neurons in the hippocampal transplants were observed within and in the vicinity of the pyramidal and granule cell layer. In neocortical transplants, PARV-immunoreactive cells were distributed in all parts of the transplant with dendrites extending in various directions. In both hippocampal and neocortical transplants, immunoreactive dendrites were smooth and displayed the characteristic regular varicosities known from in situ studies of these cells. Numerous unlabeled terminals as well as a few immunoreactive boutons established synapses on the immunoreactive dendrites. PARV-positive terminals formed the typical pericellular baskets around the immunonegative cell bodies of pyramidal neurons and granule cells in the transplants. They established symmetric synapses with cell bodies and proximal dendrites. Synapses on axon initial segments were absent or rare. Our results demonstrate that allocortical as well as neocortical nonpyramidal neurons transplanted to the neocortex of adult recipients survive transplantation, express the calcium-binding protein parvalbumin, and develop a cell-specific morphology.


Intrinsic and commissural connections of the rat inferior colliculus.

This study analyzes the distribution of the intrinsic and commissural fiber plexuses originating in the central nucleus of the inferior colliculus in the rat. The anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) was injected iontophoretically at different places along the tonotopic axis of the central nucleus and visualized immunohistochemically. In coronal sections the terminal fields of axons originating at each injection site are seen to create four well-defined bands across the rostrocaudal extent of the inferior colliculus, two in the ipsilateral and two in the contralateral side. The "ipsilateral main band" extends dorsomedially and ventrolaterally from the injection site, in register with the known isofrequency contours of the central nucleus, spanning this nucleus and extending into the dorsal cortex of the inferior colliculus. The "ipsilateral external band" is located in the external cortex, where it is oriented dorsoventrally, slightly oblique to the pial surface. In caudal sections, the ventral portion of these two bands appear to join. The two bands in the contralateral inferior colliculus occupy a symmetric position to those of the ipsilateral side, forming a mirror-like image. The position of the four bands changes as the position of the injection site is varied along the frequency gradient axis of the central nucleus. After ventromedial (high frequency area) injections, the main band is ventral and medial, and the external band ventral and lateral. After more dorsolateral (lower frequency) injections, the main band is more dorsal and lateral, whereas the external band is more dorsal but more medial. Thus, the change in the position of the external band is separate and opposite to that of the main band. We suggest that the main bands represent isofrequency contours. Since the projection from the central nucleus to the external cortex of the inferior colliculus also appears to be tonotopic, we also propose a tonotopic organization for the external cortex. The main bands overlap the terminal field of the lemniscal fibers in the central nucleus; thus, it is concluded that the intracollicular fibers contribute to the formation of the known fibrodendritic laminae of the central nucleus. A possible role in preservation of frequency information and integration of other different acoustic parameters is proposed for the main bands. The external bands could participate in polysensory integration, and the commissural connections could be involved in hitherto unknown stages of binaural processing of sound. Based on our results, several modifications are proposed for delineating the subdivisions of the inferior colliculus.


Problems with paradigms in a caring profession.

The terms 'paradigms' and 'paradigm shifts' entered everyday language following the publication in 1962 of Kuhn's book The Structure of Scientific Revolutions. Kuhn's original usage of these terms in relation to 'normal' science has since been inappropriately applied to other areas of formal knowledge. Their use in relation to nursing is conceptually flawed, and is an unnecessary distraction from the more modest, but nevertheless rigorous, approaches to the elucidation and generation of knowledge for practice which are required.


In the eye of the beholder: pretesting the effectiveness of health education materials.

Health educators at the University of Massachusetts evaluated five posters to determine whether students comprehended, identified with, and were motivated by the messages, which dealt with aspects of safer sex, alcohol use, and stress management. Pretesting, they suggest, provides an opportunity for remedying design flaws before distributing materials, improving effectiveness, and saving money by assuring that the target audience receives the intended message. Six suggestions for improving effectiveness and acceptability of printed health education materials are offered.


Sulfated glycans directly interact with mouse Thy-1 and negatively regulate Thy-1-mediated adhesion of thymocytes to thymic epithelial cells.

Thy-1 is a major brain cell surface glycoprotein of adult mammal species also expressed in rodent thymus. Despite extensive studies, the function(s) of this molecule has remained so far ill defined. We have recently shown that Thy-1 was involved in the adhesion of mouse thymocytes to thymic epithelium through a specific interaction with a heterophilic ligand(s) expressed on the epithelial cell surface. In the present study, we aimed at evaluating the interaction of sulfated glycans with mouse Thy-1, as well as its consequence on Thy-1-mediated thymic lympho-epithelial cell interaction. It was shown that 125I-labeled Thy-1 directly bound to immobilized heparin. Sulfated glycans such as pentosan sulfate, dextran sulfate, and fucoidan were found to strongly inhibit the binding of Thy-1 to heparin. In contrast, chondroitin sulfate, keratan sulfate, and heparan sulfate were not inhibitory. Sulfated glycans (e.g., pentosan sulfate, assayed at a concentration of 50 micrograms/ml) completely blocked the Thy-1-dependent adhesion of T cells to a mouse thymic epithelial cell monolayer. To explore the mechanism of this inhibition, we compared the ability of T cell to adhere to mouse thymic epithelial cell monolayer or to sulfated glycans. Our results suggest that sulfated glycans bind to a Thy-1 site distinct from that with which this molecule interacts with its heterophilic ligand. Moreover, sulfate glycans could modulate the binding of rat mAb directed at spatially distinct Thy-1 epitopes. The present results identified a potential mechanism regulating Thy-1-mediated lympho-epithelial cell adhesion.


HIV induces IL-6 production by human B lymphocytes. Role of IL-4.

In vitro, normal B cells can produce TNF-alpha and IL-6 when activated with a first signal, and cytokines and B lymphocytes from some HIV-infected individuals spontaneously secrete TNF-alpha and IL-6, although the direct involvement of HIV has not been fully explored. In this study, we examined the effects of HIV (purified virus and a recombinant envelope protein) and various IL on TNF-alpha and IL-6 in vitro production by highly purified normal B cells. HIV alone did not induce IL-6 or TNF-alpha production by B cells from healthy subjects. HIV induced IL-6 production (500 to 1500 pg) in the presence of IL-4, with a slight production of TNF-alpha. IL-6 production occurred independently of the presence or absence of TNF-alpha in contrast with Staphylococcus aureus cowan + IL-2-activated B cells. Other IL, particularly IL-2, were unable to induce IL-6 secretion by HIV-activated B cells. In vivo-activated B cells from HIV-infected patients spontaneously produce moderate quantities of IL-6 and TNF-alpha. This secretion was markedly increased by HIV, suggesting that IL-6-secreting B cells contain anti-HIV antibody-producing B cells. However, contrary to normal B cells, IL-6 production by B cells from HIV-infected patients was not further enhanced by IL-4. Then HIV itself is able to induce an autocrine production of IL-6 upon interaction with IL-4, which can contribute to the hypergammaglobulinemia and to the global B cell dysfunction observed in HIV-infected patients.


Prevention and therapy of experimental autoimmune neuritis by an antibody against T cell receptors-alpha/beta.

The mAb R73 directed to the TCR-alpha/beta of rat lymphocytes was tested for its therapeutic potential during the effector phase of experimental autoimmune neuritis (EAN) in Lewis rats. EAN can be actively induced by immunization with bovine peripheral nerve myelin, bovine P2 protein, or a peptide containing its neuritogenic epitope and serves as a model of the human Guilain-Barré syndrome. Adoptive transfer of activated P2-specific T lymphocytes also produces the monophasic disease (AT-EAN) characterized by inflammation and demyelination of peripheral nerves and highlights the central role of T lymphocytes in the pathogenesis of EAN. A single administration of the mAb R73 immediately after injection of activated P2-specific T line cells completely prevented the development of clinical and electrophysiologic signs of EAN in most animals and greatly alleviated the disease in the others. In further experiments mAb R73 was applied after the appearance of first clinical signs of EAN actively induced by immunization with a neuritogenic peptide or bovine peripheral nerve myelin. In both cases the anti-TCR-alpha/beta mAb reversed clinical signs of EAN and prevented the development of peripheral nerve dysfunction. In vivo and in vitro data suggest that impairment of Ag recognition and T cell function by occupancy of the TCR and R73-induced TCR-modulation rather than depletion of TCR-alpha/beta-bearing lymphocytes is the decisive mechanism underlying suppression of EAN that is apparent already within 48 h of the first R73 injection.


Experimental allergic encephalomyelitis mediated by cloned T cells specific for a synthetic peptide of myelin proteolipid protein. Fine specificity and T cell receptor V beta usage.

Proteolipid protein (PLP) is the major protein of central nervous system myelin. SJL (H-2s) mice immunized with a synthetic peptide corresponding to PLP residues 139-151 develop acute EAE. In this study, 6 IAs-restricted, CD4+, TCR alpha beta-bearing T cell clones were derived from SJL/J mice after immunization with this synthetic peptide. The clones responded in in vitro proliferative assays to the whole PLP molecule and to PLP peptide 139-151, but not to irrelevant Ag. They also responded to truncated and overlapping forms of the peptide but five distinct reactivity patterns were observed using these peptides. A panel of anti-TCR V beta mAb and TCR V beta-specific cDNA probes were used to determine the TCR V beta usage of the clones. Five clones were found to use four different V beta (V beta 2, V beta 6, V beta 10, or V beta 17a), whereas the V beta on the sixth clone could not be identified. Five of the clones induced EAE of varying severity upon adoptive transfer into naive syngeneic mice or mice pretreated with irradiation and pertussis and one clone was nonencephalitogenic. The Ag-specific proliferative response of all but the nonencephalitogenic clone could be blocked by an anti-CD4 mAb. Thus, the clones showed differences in their fine specifity, TCR V beta usage, sensitivity to antibody blocking, and encephalitogenic potency. These data demonstrate that the T cell response to the encephalitogenic PLP peptide 139-151 is heterogeneous.


Differences in activation of normal and glycosylphosphatidylinositol-negative lymphocytes derived from patients with paroxysmal nocturnal hemoglobinuria.

In these studies, the role of glycosylphosphatidylinositol (GPI)-anchored surface molecules during T cell activation was investigated in fresh T cells and T cell lines obtained from patients with paroxysmal nocturnal hemoglobinuria. For control, GPI-expressing T cells of the same patients were used. Unstimulated GPI- T cells exhibited significantly reduced surface expression of the activation Ag CD45R0, compared with GPI+ T cells. In addition, in measurements of proliferation, IFN-gamma production, and induction of second messengers such as cytoplasmic Ca2+, CD48- lymphocytes showed a similar response to TCR-specific stimulation, compared with CD48+ lymphocytes. In contrast, stimulation with the lectin PHA produced a decreased response of CD48- lymphocytes in these functions. In addition, stimulation with cross-linked CD59 mAb increased the proliferation of GPI-molecule expressing CD48+ T cell lines only. From these data, it can be concluded that GPI-anchored surface molecules play an important role in T lymphocyte activation.


A monoclonal anti-idiotype specific for human polyclonal IgM rheumatoid factor.

One of the hallmarks of rheumatoid arthritis (RA) is the production of high titers of rheumatoid factor (RF) antibody directed against the Fc portion of IgG. Anti-Id that recognize the majority of monoclonal RF from patients with B cell dyscrasias are reactive with only 1 to 2% of these polyclonal RF from RA patients. We describe a new monoclonal anti-Id, 4C9, that recognizes a L chain determinant on polyclonal IgM RF from patients with RA but does not recognize a panel of monoclonal RF from patients with B cell malignancies. 4C9 reactivity is found in the serum of 34/43 RF-positive RA patients and in 12/12 RF-positive synovial fluids, but in only 1/14 RF-negative sera from RA patients and 1/22 sera containing monoclonal IgM RF. 4C9 reactivity is highly enriched in purified IgM RF from nine RA patients and represents a variable percentage of total IgM RF up to a maximum of 23%. Furthermore, 4C9 reactivity is enriched in the synovial fluid of three of five RA patients compared with serum, suggesting that 4C9-reactive IgM RF are synthesized within the joint. IgG RF from RA synovial fluids are not 4C9 reactive, indicating either that different genes are used to encode IgM and IgG RF in RA patients, or that IgG RF have somatically mutated away from idiotypic reactivity.


Membrane IgM-mediated signaling of human B cells. Effect of increased ligand binding site valency on the affinity and concentration requirements for inducing diverse stages of activation.

The potential for ligand-initiated signal transduction through B cell membrane IgM is assessed in terms of ligand concentration, binding site valency, and binding site affinity for membrane Ig. Estimates of the physicochemical requirements for achieving G0* enhancement of class II MHC expression, G1 entry, and S phase entry in human B cells were made by comparing the stimulatory effects of three affinity-diverse anti-Cmu2 mAb when in bivalent (unconjugated) form, or as mAb-dextran conjugates with low binding site valency (oligovalent ligands) or high binding site valency (multivalent ligands). An increase in binding site number (and concomitant molecular mass) caused a profound reduction in both the minimal concentration and affinity requisites for B cell activation. The enhancing effect of increased binding site valency was most evident for the signaling of those most distal stages in B cell activation, i.e., G1 and S phase, which were difficult to induce with bivalent ligands. The results suggest that highly multimeric TI-2 Ag may be good immunogens because they are able to elicit a full activation response not only from infrequent high affinity B cells, but also from a substantial proportion of the many lower affinity Ag-specific B cells in virgin B cell populations. Interestingly, the activation of B cells by ligands with binding sites of high intrinsic affinity (Ka = 5 x 10(8) M-1) was less influenced by increases in binding site valency than was B cell activation by ligands with intermediate binding site affinity (Ka = 2 x 10(7) M-1). This suggests that the minimal epitope valency requirement for T cell-independent B cell activation by mIg cross-linking Ag may be dependent on the intrinsic affinity with which membrane Ig molecules on a given B cell interact with the redundantly expressed epitopes.


Anti-human IgG causes basophil histamine release by acting on IgG-IgE complexes bound to IgE receptors.

We have reexamined the ability of anti-human IgG antibodies to induce histamine release from human basophils. A panel of purified murine mAbs with International Union of Immunological Societies-documented specificity for each of the four subclasses of human IgG was used. Of the 24 allergic subjects studied, the basophils of 75% (18/24) released greater than 10% histamine to one or more anti-IgG1-4 mAb, whereas none of the 13 nonatopic donor's basophils released histamine after stimulation with optimal amounts of anti-IgG mAb. The basophils of 85% (11/13) of the nonatopic donors did respond to anti-IgE challenge, as did 92% (22/24) of the atopic donor cells. Histamine release was induced most frequently by anti-IgG3, and 10/18 anti-IgG responder cells released histamine with mAb specific for two or more different subclass specificities. The rank order for induction of histamine release was anti-IgG3 greater than anti-IgG2 greater than IgG1 greater than anti-IgG4. As in our previous study using polyclonal anti-IgG, 100- to 300-micrograms/ml quantities of the anti-IgG mAb were required for maximal histamine release, about 1000-fold higher than those for comparable release with anti-human IgE. Specificity studies using both immunoassays and inhibition studies with IgE myeloma protein indicated that anti-IgG induced histamine release was not caused by cross-reactivity with IgE. Ig receptors were opened by lactic acid treatment so that the cells could be passively sensitized. Neither IgE myeloma nor IgG myeloma (up to 15 mg/ml) proteins could restore the response to anti-IgG mAb. However, sera from individuals with leukocytes that released histamine upon challenge with anti-IgG mAb could passively sensitize acid-treated leukocytes from both anti-IgG responder and nonresponder donors for an anti-IgG response. The only anti-IgG mAb that induced release from these passively sensitized cells were those to which the serum donor was responsive. Sera from non-IgG responders could not restore an anti-IgG response. These data led to the hypothesis that the IgG specific mAb were binding to IgG-IgE complexes that were attached to the basophil through IgE bound to the IgE receptor. This was shown to be correct because passive sensitization to anti-IgG could be blocked by previous exposure of the basophils to IgE. We conclude that anti-IgG-induced release occurs as a result of binding to IgG anti-IgE antibodies and cross-linking of the IgE receptors on basophils.


A universal T cell epitope-containing peptide from hepatitis B surface antigen can enhance antibody specific for HIV gp120.

Peptide-based vaccines that directly target T cell or B cell epitopes may have significant advantages over conventional vaccines. Further, synthetic chimeric peptides that combine strong T cell epitopes with poorly immunogenic, but immunodominant, B cell epitopes or strain-conserved B cell epitopes may be useful in eliciting antibody to such important regions. Here we characterize a human T cell epitope analyzed in 54 individuals immunized with a hepatitis B virus surface Ag vaccine. Primary cultures from a total of 59 immunized donors were assessed for their ability to respond to hepatitis B virus surface Ag and peptides, and five were non-responders (8.5%). T cell lines were established from the remaining 54 responders. Of the responders, it was found that the peptide representing amino acids 19 through 33 (19-33) elicited significant proliferation in lines derived from 50 donors. This "universal" T cell epitope, which was recognized in donors of many different HLA-DR and -DQ haplotypes, was then used to construct a chimeric peptide containing 19-33 and the third V region loop structure (V3 loop) of HIV-1 envelope gp 120, in an attempt to augment the immune response to the V3 loop peptide. The V3 loop is the region to which significant neutralizing antibody is directed. Thus, a strong immune response to a synthetic peptide that contains the strain-conserved V3 loop region could have significant therapeutic implications. The V3 loop/19-33 peptide was then used to prime mice, to determine whether V3 loop-specific antibody could be induced. The peptide elicited potent 19-33-specific proliferation in T cells isolated from draining lymph nodes, and in six of six mice anti-V3 loop antibody was elicited. Further, V3 loop/19-33-primed animals made significant levels of antibody that bound rgp120. These data suggest that, when a major T cell epitope is synthesized in tandem with the V3 loop, a significant immune response against the loop can be elicited. Thus, given the finding that neutralizing antibody may play a role in the control and/or prevention of HIV infection, an HIV vaccine composed of a T cell epitope-containing peptide may prove effective. In addition, this type of approach can be generalized to the design of peptide-based vaccines.


Human recognition of T cell epitopes on the Plasmodium vivax circumsporozoite protein.

In order to identify T cell epitopes recognized by human in the Plasmodium vivax circumsporozoite protein, 28 overlapping synthetic peptides spanning the entire circumsporozoite protein were tested for their ability to stimulate proliferation of PBMC from 22 adults living in a malaria-endemic area of the Colombian Pacific Coast and from four individuals who never had a history of malaria infection. In addition, BALB/c mice were immunized with pools of peptides, and their lymph node cells were stimulated in vitro with individual peptides. Four epitopes were recognized by human lymphocytes but not all of them by mice. One of the epitopes was located inside the central repetitive B cell immunodominant domain. Several of the variants of the repeats were recognized by about one-third of the studied individuals. Another T cell epitope was located in the amino terminus and the other two in the carboxyl region. Peptides were recognized by both immune and nonimmune donors. Some of them were frequently recognized suggesting a lack of genetic restriction, whereas some others were recognized by only a few individuals but induced strong proliferation. These epitopes may be of potential value for a malaria subunit vaccine.


A synthetic peptide spontaneously self-assembles to reconstruct a group-specific, conformational determinant of hepatitis B surface antigen.

A cysteine-rich peptide of sequence 124 to 147 of the major protein of hepatitis B surface Ag (HBsAg) was synthesized. On cleavage and subsequent work-up it was found that all of the cysteine sulfhydryl groups had spontaneously formed disulfide bonds to yield a heterogenous mixture of multiple forms with molecular masses ranging from 8 to 35 kDa (peptide OS[124-147]). In a direct ELISA peptide OS[124-147] showed a high degree of cross-reactivity with polyclonal anti-HBsAg antiserum whereas the HBsAg-related antigenicity of its disulfide-reduced analogs was insignificant. Peptide OS[124-147] was also recognized by all 15 of the anti-HBsAg-positive human sera tested. Further studies revealed that peptide OS[124-147] represents the conformational, disulfide-dependent "a" determinant of HBsAg and elicits antibodies that cross-react with a variety of HBsAg subtypes. Anti-peptide antibodies bound to the corresponding native epitope with an apparent affinity higher than that of homologous antisera. Finally, polyclonal anti-OS[124-147] antibodies could also immunoprecipitate purified Dane particles in solution. Together these studies indicate that peptide OS[124-147] represents an excellent candidate component of a peptide-based vaccine for hepatitis B.


Unique palindromic sequences in synthetic oligonucleotides are required to induce IFN [correction of INF] and augment IFN-mediated [correction of INF] natural killer activity.

Thirty-mer single-stranded oligonucleotides, with a sequence chosen from the known cDNA encoding the 64-kDa protein named Ag A or the MPB-70 protein of Mycobacterium bovis BCG and the human cellular proteins such as complement component 1 inhibitor and Ig rearranged lambda-chain, were used to dissect the capability to induce IFN and to augment NK cell activity of mouse spleen cells by coincubation in vitro. Three with the hexamer palindromic sequence as GACGTC were active, whereas two kinds of oligonucleotides with no palindrome were inactive. The oligonucleotides containing at least one of the different palindromic sequences showed no activity. When a portion of the sequence of the inactive oligonucleotides was substituted with either palindromic sequence of GACGTC, AGCGCT, or AACGTT, the oligonucleotide acquired the ability to augment NK activity. In contrast, the oligonucleotides substituted with another palindromic sequence such as ACCGGT was without effect. Furthermore, exchange of two neighboring mononucleotides within, but not outside, the active palindromic sequence destroyed the ability of the oligonucleotides to augment NK cell activity. Stimulation of spleen cells with the substituted oligonucleotide, A4a-AAC, induced production of significant amounts of IFN-alpha/beta and small amounts of IFN-gamma. Augmentation of NK activity of the cells by the oligonucleotide was ascribed to IFN-alpha/beta production. These results strongly suggest that the presence of the unique palindromic sequences, such as GACGTC, AGCGCT, and AACGTT, but not ACCGGT, is essential for the immunostimulatory activity of oligonucleotides.


Surface markers, heavy chain sequences and B cell lineages.

A unifying theory of B cell development and lineage commitment is presented. There are two firmly established B lineages: cells which normally arise only from fetal sources and lack N insertions in their rearranged heavy chains; and N-containing cells which arise from adult bone marrow precursors (and perhaps from late fetal sources). Commitment to the expression of CD5 and the capacity for long-life (or self-renewal) are induced as a consequence of sIg cross-linking, typically by a repeating epitope, thymus independent type two antigen. Alternatively, activation resulting from cognate interaction with a helper T cell does not induce CD5 but results in lower expression of J11d. In this case activation occurs in the absence of sIg cross-linking. It is further proposed that differences in the Ig repertoire make it highly likely that fetal/neonatal, but not adult derived B cells will be induced to express CD5. The model offers a plausible explanation for the correlation of CD5 expression and natural autoantibody production by neonatal B cells. Possible sources of pathogenic autoantibody are discussed in the context of this model.