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We have employed both protein chemical and molecular biological approaches to determine the ligand binding domain of the endothelin-B subtype (ETB) receptor. The human ETB receptor purified from human placenta by using affinity chromatography was cross-linked with 125I-labeled endothelin-1 (ET-1) and then incubated in the presence of trypsin or thermolysin under nondenaturing conditions. The N-terminal amino acid sequence of the radiolabeled polypeptide encompassed approximately 115 amino acid residues starting from Ile85 of the human ETB receptor. This was confirmed by experiments in which the binding activity of endothelin-1 to various chimeric endothelin receptors was monitored in the presence and absence of competitive endothelin receptor antagonists such as BQ-123 and bosentan. The region from Ile138 to Ile197 (60 amino acid residues) of the ETB receptor was found to interact with both antagonists. Therefore, this sequence was determined to be the ligand binding domain. In addition, we found that part of the N-terminal domain in close proximity to the first transmembrane region was required for the ligand binding activity of the ETB receptor, and the 12 amino acid residues from Ser390 to Leu401 at the proximal cytoplasmic tail are perhaps necessary to maintain the ligand binding site in active form. The cysteine rich region from residue 400 to residue 403 in the C-terminus of the ETB receptor is involved in coupling of the guanine nucleotide-binding regulatory protein for ET-1-induced signal transduction.

Background: The optimal management of patients with brain tumors requires knowledge of the tumor characteristics upon presentation and the discovery of recurrence after therapy. Thallium-201 (Tl-201) chloride has shown varying uptake in tumors, depending on their viability and the type and degree of malignancy. This study explores the diagnostic potential of thallium imaging in patients with brain tumors., Methods: Forty-three Tl-201 single photon emission computed tomographic scintigrams were performed on 40 patients with intracranial neoplasms, nearly equally divided between patients with no prior treatment and patients who had prior treatment and were suspected to have recurrent tumor and/or radiation necrosis. A thallium tumor index was calculated as the ratio of counts for a region of interest drawn in the lesion area and its mirror image in normal brain tissue. A two-tailed Student's t test was performed to compare the thallium index and histopathologic findings., Results: A value of 1.5 for the thallium tumor index allowed for the best correlation between the prediction of malignancy and the histopathologic results. In the pretreatment group, a thallium tumor index greater than 1.5 correlated with high grade malignancy, and less than 1.5 correlated with either a well differentiated astrocytoma or benign cyst. In the posttreatment group, a thallium tumor index greater than 1.5 correlated with recurrent and/or residual malignant tumor., Conclusions: For those patients undergoing initial evaluation, the thallium study can help in the differential diagnosis of an intracranial mass lesion and offers confirmation of results of biopsy. For those patients who already have received treatment, the study can be used to detect recurrent or residual tumor.

Bosentan (Ro 47-0203, 4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2- methoxy-phenoxy)-2,2'-bipyrimidin-4-yl]-benzenesulfonamide) is a new non-peptidic mixed antagonist of endothelin receptors whose binding activity was two orders higher for the endothelin ETA receptor than that for the endothelin ETB receptor. To identify which region of the human endothelin ETA receptor interacts with bosentan, we created various chimeric endothelin receptors containing domains from the endothelin ETA and ETB receptors in Chinese hamster ovary cells and studied the effect of bosentan on the binding of endothelin-1 to the chimeric receptors. We found that the chimeric endothelin ETB receptor containing domains from the endothelin ETA receptor, the second extracellular region including the proximal transmembrane region (B-region) revealed an affinity toward bosentan which was similar to that of the endothelin ETA receptor. In contrast, the chimeric endothelin ETA receptor, containing the B-region of the endothelin ETB receptor, reduced the binding affinity to the level of the endothelin ETB receptor. Since bosentan competes with endothelin-1 for binding to the endothelin ETA receptor, this receptor antagonist seems to interact with the (140-144) KLLAG sequence located at the carboxylterminus of the second transmembrane region of the endothelin ETA receptor, required for the natural ligand binding.

To investigate the endothelin-3 (ET-3) binding region of the endothelin-B (ETB) receptor, we have transiently produced various chimeric endothelin receptors in transfected Chinese hamster ovary cells. Using 125I-ET-1 as the radioactive ligand in the displacement experiment, the replacement of both the second and third extracellular regions including the flanking transmembranes of the ETB receptor with the corresponding domains of the endothelin-A (ETA) receptor, increased the apparent Ki value for ET-3 from 5 x 10(-11) M to 10(-8) M. The affinity of this chimeric receptor, ETB-BC, for ET-3 was about two orders lower than ETB yet one order higher than ETA. Previously we have reported the involvement of Lys-140 located in the C-terminus of the second transmembrane region of the ETA receptor for ET-1 binding (Eur. J. Biochem., 220, 37-43, 1994). To assess the importance of the corresponding Lys-161 of the ETB receptor in ET-3 binding, we have replaced it with Ile in the ETB receptor. The mutant receptor had a 5.6-fold reduction in its affinity for ET-3, but its affinity for ET-1 remained similar. These results demonstrate that Lys-161 of the receptor is important for high affinity binding with ET-3 which, in part, confers the non-selective binding characteristics of the ETB receptor for ET isopeptides.

From September 1983 through January 1989, 8 patients with a diagnosis of squamous cell carcinoma of the esophagus AJCC stages I to III, were treated with intracavitary brachytherapy following external irradiation. Within 3 months after completion of treatment, there was radiographic evidence of complete response in 6 of the 8 patients. None of the responders recurred locally. All the patients have died, with a mean survival of 17 months. One patient died of a stroke 2 1/2 years after completion of treatments without clinical evidence of esophageal cancer. Treatment was well tolerated, with minimal acute toxicity, and one patient developed stricture at the site of tumor, which was successfully treated by dilatation. Addition of systemic chemotherapy to this regimen warrants investigation.

To investigate the nuclear signalling pathway induced by endothelin (ET) isopeptides, we have established permanent Chinese hamster ovary (CHO) cell lines, CHO-ETA/fos-lacZ and CHO-ETB/fos-lacZ, that produce both a c-fos-beta-galactosidase fusion protein and either the type A or the type B human ET receptor. These cell lines permitted a colorimetric measurement of c-fos expression, which was induced by the signal transduction system with ET receptors and ET isopeptides. We found that the ET-1-dependent c-fos expression was so efficient that it could respond to low concentrations (even a physiological concentration) of ET-1. For example, CHO-ETA/fos-lacZ and CHO-ETB/fos-lacZ responded to ET concentrations of 5 x 10(-9) M and 5 x 10(-13) M respectively. Using this highly sensitive system, the H-7 sensitive protein kinase was found to be involved in signal transduction mediated by ETA, and also partly in the ETB-mediated pathway. These lines of evidence suggest that c-fos expression occurs through at least two different pathways, depending on the concentration of ET in plasma.

A gene whose overexpression can endow Saccharomyces cerevisiae cells with resistance to HM-1 killer toxin was cloned from an S. cerevisiae genomic library. This gene, designated HKR1 (Hansenula mrakii killer toxin-resistant gene 1), contains a 5.4-kb open reading frame. The predicted amino acid sequence of the protein specified by HKR1 indicates that the protein consists of 1,802 amino acids and is very rich in serine and threonine, which could serve as O-glycosylation sites. The protein also contains two hydrophobic domains at the N-terminal end and in the C-terminal half, which could function as a signal peptide and transmembrane domain, respectively. Hkr1p is found to contain an EF hand motif of the calcium-binding consensus sequence in the C-terminal cytoplasmic domain. Thus, Hkr1p is expected to be a calcium-binding, glycosylated type I membrane protein. Southern and Northern (RNA) analyses demonstrated that there is a single copy of the HKR1 gene in the S. cerevisiae genome, and the transcriptional level of HKR1 is extremely low. Gene disruption followed by tetrad analysis showed that HKR1 is an essential gene. Overexpression of the truncated HKR1 encoding the C-terminal half of Hkr1p made the cells more resistant to HM-1 killer toxin than the full-length HKR1 did, demonstrating that the C-terminal half of Hkr1p is essential for overcoming the effect of HM-1 killer toxin. Furthermore, overexpression of HKR1 increased the beta-glucan content in the cell wall without affecting in vitro beta-glucan synthase activity, suggesting that HKR1 regulates beta-glucan synthesis in vivo.

To investigate the ligand-binding site of the human endothelin-A-receptor subtype (ETA), we have produced various chimeric and mutated receptors in chinese hamster ovary cells. The substitution of Lys140 with Ile located in the C-terminus of the second transmembrane region caused a 13-fold reduction in affinity for endothelin-1 (ET-1) and 3.6-fold lower Bmax than those values for the original receptor. Correspondingly, the mutated ETA receptor with the Lys140-->Ile substitution failed to induce an increase in the intracellular calcium concentration in the presence of 1 nM ET-1. Thus, the Lys140 in the ETA receptor is important in ligand binding. ETA and ETB receptors possess the ET isopeptides selective and non-selective binding activities, respectively. Displacement experiments and the binding of 125I-ET-3 to various chimera receptors demonstrated that both the third and fourth extracellular regions, including the flanking transmembrane regions, are responsible for the ligand-binding selectivity of the ETA receptor.

Background: The monoclonal antibody anti-epidermal growth factor receptor (EGFr) antibody-425, against the epidermal growth factor receptor, has the potential to bind specifically to gliomas and not normal brain tissue. A prospective study was conducted (1986-1988) to evaluate the use of Indium-111 (111In)-labeled anti-EGFr-425 in the localization of gliomas before radioimmunotherapy with Iodine-125 (125I)-labeled anti-EGFr-425., Methods: Twenty-eight patients with intracranial neoplasms were injected intravenously with an average dose of 2.2 mCi 111In-labeled anti-EGFr-425. Planar and single-photon emission computed tomography scans were performed after 48 and 72 hours. Control studies also were performed in two cases with 111In-labeled Co 17-1A (an antibody to colorectal cancer) and in one case with unlabeled 111In chloride., Results: The immunoscintigraphic findings were generally in good agreement with computerized tomographic findings. The definitive diagnosis was established by biopsy findings: 23 gliomas (1 Grade I, 5 Grade II, 6 Grade III, and 11 Grade IV), 1 meningioma, and 4 metastatic lesions. The localization of gliomas with 111In-labeled anti-EGF-425 had a sensitivity of 0.96, a specificity of 0.60 and an accuracy of 0.90., Conclusion: Immunoscintigraphy with 111-In labeled anti-EGFr-425 can be useful in the management of malignant gliomas, especially before radioimmunotherapy with 125I-labeled anti-EGFr-425.

Twenty-six patients (20 females, 6 males) with localized tumors of the pelvis, including 3 primary advanced (PRIM), 7 persistent (PERS), 10 recurrent (REC), and 6 metastatic (MET) tumors, were treated with a combination of low-dose rate (LDR) iridium 192 interstitial radiotherapy (IRT), interstitial 915 MHz microwave hyperthermia (IHT), and external beam radiotherapy (RT). Histological diagnoses were squamous cell carcinoma in 13 (50%), adenocarcinoma in 12 (46%) and soft tissue sarcoma in 1 (4%) lesion. Tumor sites were cervix in 8 (31%), colorectum in 6 (23%), vagina in 4 (15%), anus in 3 (12%), ovary in 2 (8%), and other sites in 3 (12%) lesions. IHT was administered immediately before iridium 192 was placed and after its removal for 45-60 minutes at 41-44 degrees C. On December 31, 1991 median follow-up was 25 months (mean: 23 months; range: 5-65 months). At 3 months follow-up (FU), complete remission (CR) occurred in 17 (65%), partial remission (PR) in 7 (27%), and no change or progressive disease (NC/PD), in 2 (8%) lesions. At 12 months FU, in 16 of 21 patients (76%) local control (LC) was achieved, with 1 (5%) patient exhibiting a slow tumor regression. After combined IRT-IHT locoregional relapse or tumor regrowth occurred in 8/26 (31%): 5 (19%) outside and 3 (12%), inside the previously treated volume; relapses occurred within 8-30 (mean: 18) months of follow-up. Factors influencing initial (3 months FU) and long-term tumor response (12 months FU) included tumor class, tumor volume, total radiation dose, and thermal parameters with "good quality of heating" (TQ 41 degrees C > or = 75%) and high minimum tumor temperature (Tmin(av) > or = 41 degrees C). Treatment toxicity was acceptable: whereas 8 (31%) patients experienced acute side effects, which subsided within weeks, 7 (27%) developed long-term complications. Thermal damage was associated with IHT treatments exceeding maximum average temperatures of > or = 44 degrees C and maximum peak temperatures of > or = 45 degrees C.

To investigate the overall prevalence of chlamydial infections in light (i.e. non-draught) horses in Japan, 599 sera obtained from 12 localities in 1991 were tested for complement fixation antibodies. The mean antibody positive rates of the all sera were 15.2% (91/599) and the regional positive rates were higher in Honshu (19.1%, 48/251) and Kyushu (20.0%, 20/100) than in Hokkaido (9.3%, 23/248). In Honshu, the highest rate (56.0%, 28/50) was observed in Utsunomiya. Analysis of the positive rate in different age groups showed that the 2-5 years age-group had the highest prevalence of chlamydial infections. This indicates that chlamydial infection is prevalent in light horses in Japan.

The carboxyl groups on the surface of latex beads were linked to amino moiety of cytidine residue of oligo(dC)10(dT)30. The resultant latex beads-(dC)10(dT)30 showed a very stable suspension and yet is precipitable to a small pellet by centrifugation. These properties merits the oligomer-linked beads to be applied for experiments in which poly(A)+ mRNAs are involved. An efficient (> 95%) hybridization to poly(A)+ mRNA occurred in a short reaction period (10 min), and more than 95% of bound mRNAs were recovered from the beads by heating (65 degrees C, 5 min) followed by centrifugation. Interestingly, the poly(A)+ mRNAs could be transcribed to cDNAs in situ by reverse transcriptase, with the covalently linked oligo(dT)30 as primers. These properties allowed the oligo(dT)30-latex to prepare the cDNA covalently bound to latex which was used for mRNA hybrid subtraction. In a model experiment with the mixture of vaccinia virus and HeLa mRNAs, about 200-fold enrichment of vaccinia mRNA species was obtained after four cycles of hybrid subtraction with HeLa cDNA-latex.

A clone containing the promoter, the exon 1 and exon 2 of human genomic ETA gene was isolated and sequenced. The transcription initiation site and TATA box were identified at 528 bp upstream of the initiation codon and 24 bp upstream of the transcription initiation site, respectively. The 534 bp fragment containing 5'-flanking sequence and most of the exon 1 of the human ETA gene showed a promoter activity corresponding to 55% of SV40 early promoter activity when placed upstream of the luciferase gene and transfected into CHO cells. Thus, the TATA box which consists of the TAAAAA sequence is functional for transcription of the human ETA gene. The coding region of the human ETA receptor, therefore, starts from exon 2 which contained the first and the second transmembrane regions. The human ETA gene was demonstrated to be a single pair of copy by Southern blot analysis, and it was localized in chromosome 4 by analysis with a panel of human-rodent somatic cell hybrid lines.

The ligand binding site to the ETA receptor was investigated by substitution of each 5-amino acid sequence located in the second extracellular (B) region of the ETA receptor with the cognate sequences of the beta 2-adrenergic receptor. A 5-amino acid sequence (140-KLLAG-144) in the B-loop region was implicated as the most important element required for ligand binding. In addition, both the third and the fourth extracellular regions (C- and D-loops), including the flanking transmembrane regions, were found to play an important role in ligand selection. As for the biological significance of the intracellular regions of the ETA receptor, we have found that the C-terminal 8-amino acid residues located in close proximity to the seventh transmembrane region and the C-terminal 16-amino acid residues in the third intracellular loop are important for the binding of ET-1. Therefore, the intracellular third loop and C-terminal domains seem to contribute to the three-dimensional structure of the ligand binding site located in the extracellular domains. The same lines of experiment showed that the ETA receptor requires > 13 amino acid residues at the proximal cytoplasmic tail and 10 amino acid residues in the C-terminal region of the third intracellular loop to induce an ET-1-dependent increase in [Ca2+]i. Both regions are possibly involved in the interaction with G-protein.

We have investigated the function of the C-terminal and the third intracellular domains of the ETA receptor by expressing truncated and mutated ETA receptors in COS-7 and CHO cells. All the C-terminal truncated ETA receptors were produced at a similar expression level and were detected in the cell membrane using indirect immunostaining. The sizes of the truncated ETA receptors were decreased in proportion to the molecular mass of the truncated amino acid sequence. When the ligand binding activities were determined for various truncated ETA receptors, it was found that more than eight amino acid residues at the proximal cytoplasmic tail of the ETA receptor were required for ET-1 binding. In addition, the deletion of 16 C-terminal amino acid residues from the third intracellular loop severely decreased the ligand binding activity. It seems that deletion of these cytoplasmic domains of the ETA receptor influences the three-dimensional structure of the ligand binding site located in the extracellular domains. The ETA receptor required more than 13 amino acid residues in the proximity of C-terminal cytoplasmic tail and 10 amino acid residues in the C-terminal region of the third intracellular loop to induce the ET-1 dependent increase in intracellular calcium concentration. Both regions are possibly coupled with G-protein to transmit the ET-1 signal.

We have developed a novel method for constructing NotI linking and boundary libraries using a modified "solid-supported ligation primer" (restriction trapper). The restriction trapper could be used to purify the DNA fragments with a specific restriction enzyme cutting site(s) at their ends. The method uses a ligation and recutting reaction with double-stranded DNA ends of a hairpin-shaped oligolinker which is connected covalently to the surface of the latex beads. Selectivity is based on the specificity of the restriction enzyme for its recognition site, resulting in efficient purification. We applied this technique to the construction of high-quality NotI linking and NotI boundary libraries, which contain almost all the NotI sites of the genome and, in addition, are free of illegitimately ligated clones.

Various chimeric ETA and ETB receptors were produced in CHO cells for the elucidation of a specific domain which influences the affinity of the receptor toward BQ-123, a selective ETA antagonist. Replacement of the first extracellular loop domain (B-loop) of the ETA receptor with the corresponding domain of the ETB receptor, reduced the inhibition by BQ-123 drastically, while the replacements of other extracellular domains of ETA did not. By contrast, the introduction of the B-loop of ETA in place of the corresponding domain of the ETB receptor endowed the ETB-based chimeric receptor with a sensitivity to BQ-123. These observations suggest that the B-loop domain of the ETA receptor is involved in ligand binding.

We have investigated the function of N-terminal and C-terminal domains of the human ETA receptor by expressing truncated mutants in COS-7 cells. Three kinds of ETA receptors truncated in the N-terminal extracellular or C-terminal intracellular domains were produced. Deletion of the entire extracellular N-terminal or intracellular C-terminal domain completely inactivated the ET-1 binding activity. However, the deletion of one half of the N-terminal extracellular domain of the ETA receptor, missing one of two N-linked glycosylation sites, maintained complete binding activity. Specific monoclonal antibodies detected all the truncated ETA receptors in the cell membrane fraction of transfected COS-7 cells. The size of the ETA receptor was heterogeneous due to differential glycosylation and distributed in 48K, 45K and 42K dalton bands in Western blot analysis. These results demonstrated that a part of the N-terminal domain in close proximity to the first transmembrane region is required for the ligand binding activity of the ETA receptor, and the C-terminal domain is perhaps necessary as an anchor for maintenance of the binding site.

The cloned cDNA genes for endothelin receptors ETA and ETB were expressed in COS cells, and the binding characteristics of the two receptors with three isopeptide ligands (ET-1, ET-2, and ET-3) were examined in detail. The results indicated that the stability of receptor-ET-1 complexes formed with ETA and ETB were significantly different from each other, while their affinities to ET-1 were similar. The preformed ETA-ET-1 complex readily dissociated upon SDS-PAGE, as did many of the other receptors so far studied, while the ETB-ET-1 complex survived SDS-PAGE when it was run at low temperature (approximately 4 degrees C). Clear differences in stability were also shown in comparative studies of acid treatment of the two types of complexes. Only the ETB-ET-1 complex was resistant to acid treatment (0.2 M acetic acid, 0.5 M NaCl), and its 50 kDa monomeric receptor-ligand complex remained intact. The ETB-ET-1 complex (50 kDa) formed at 4 degrees C on the surface of COS cells, however, was susceptible to limited proteolysis at 37 degrees C that reduced the molecular size of the complex to a distinct 35 kDa. No such size reduction was observed with the preformed ETA-ET-1 complex. The overall structure of two endothelin receptors, as deduced from the sequence of cloned cDNAs, is similar in many respects. However, the present findings demonstrate distinct differences in the biochemical nature of the two receptors, which suggest their distinct biological functions.

It is a well-known fact among clinicians that sunlight may exacerbate atopic dermatitis (AD), but little is known beyond that. In a preliminary study investigating this phenomenon, 19 patients with AD were selected for phototests. All of them had a normal minimal erythema dose (MED). However, 3 patients (15.7%) demonstrated abnormal cutaneous responses 24-72 h after provocation with ultraviolet light B (UVB). None of the patients had a positive response to pure ultraviolet light A (UVA) irradiation of up to 9 J/cm2. The photobiological results of this study confirm the existence of photosensitivity in AD and indicate that UVB wavelengths are responsible for it.

Twenty-five patients with primary presentation of malignant astrocytoma, astrocytoma with anaplastic foci, and glioblastoma multiforme were treated with surgical resection and definitive radiation therapy followed by intravenous or intra-arterial administration of Iodine-125 labeled monoclonal antibody-425, which binds specifically to human epidermal growth factor receptor. The patients presented with primary untreated disease, positive contrast enhanced computed tomography scans of the brain, and compatible clinical symptoms. In this Phase II clinical trial, the patients had surgical debulking or biopsy followed by definitively administered external beam radiation therapy and one or multiple doses (35 to 90 mCi per infusion) of radiolabeled antibody. The total cumulative doses ranged from 40 to 224 mCi. The administrations of the radiolabeled antibody were performed in most cases 4-6 weeks following completion of the primary surgery and radiation therapy. Ten patients had astrocytoma with anaplastic foci and 15 had glioblastoma multiforme. No significant life-threatening toxicities were observed during this trial. At 1 year 60% of the patients with astrocytoma with anaplastic foci or glioblastoma multiforme are alive. The median survival for both groups was 15.6 months.

We isolated a new calcium-binding protein from porcine cardiac muscle by calcium-dependent hydrophobic and dye-affinity chromatography. It showed an apparent molecular weight of 11,000 on SDS-PAGE. Amino acid sequence determination revealed that the protein contained two calcium-binding domains of the EF-hand motif. The cDNA gene coding for this protein was cloned from the porcine lung cDNA library. Sequence analysis of the cloned cDNA showed that the protein was composed of 99 amino acid residues and its molecular weight was estimated to be 11,179. Immunological and functional characterization showed that the recombinant S100C protein expressed in Escherichia coli was identical to the natural protein. Homologies to calpactin light chain, S100 alpha and beta protein were 41.1%, 40.9% and 37.5%, respectively. The protein was expressed at high levels in lung and kidney, and low levels in liver and brain. The tissue distribution was apparently different from those of the other S100 protein family. These results indicate that this protein represents a new member of the S100 protein family, and thus we refer to it as S100C protein.

A cDNA coding for the human A-type endothelin receptor (ETA) was cloned from a human placenta cDNA library. The cDNA contained the entire coding sequence for the 427 amino acid protein with a relative Mr of 48,722. The deduced amino acid sequence of the human ETA was, respectively, 94% and 93% homologous with the sequence of bovine ETA and rat ETA, but was only 64% homologous with that of the human ETB receptor. Upon expression in COS-1 cells, the human ETA receptor showed binding activity to ETA, with the highest selectivity to ET-1. Northern blot analysis showed that the mRNA of human placenta ETA consists of one species 5 kilo-nucleotides in length, and the same analysis for the uterus, testis, heart and adrenal gland of Cynomolgus monkey showed that the cognate mRNAs are widely distributed.

We report 5 cases of photocontact dermatitis due to suprofen, a nonsteroidal anti-inflammatory drug introduced to the Japanese market in 1989, and available as a 1% ointment. The patients developed pruritic eczematous lesions after applying the ointment for from 2 weeks to 3 months. All 5 patients reacted positively to photopatch testing with ultraviolet A (UVA) and suprofen down to 0.1-0.01% pet., and 3 patients showed positive reactions with ultraviolet B (UVB) and suprofen down to 1.0-0.1%. Moreover, all patients showed a cross-reaction with tiaprofenic acid, which has a very similar chemical structure to suprofen. However, there was no cross-reaction between suprofen and ketoprofen. Prescribers should be aware of the existence of photocontact sensitivity due to these drugs.

A novel human cell line, KMT-2, from umbilical cord blood cells was established based on the selection of cultures in the presence of recombinant human interleukin-3 (IL-3) and the sorting of cells with anti-My 10 antibody. Morphologic and cytochemical studies (peroxidase negative, Sudan-black negative, chloroacetate esterase negative, PAS positive, nonspecific esterase positive) and phenotyping (HLA-DR, My7 = CD13, My9 = CD33, My10 = CD34, MCS-2, LeuM1 positive, glycophorin A negative, and P2 negative) suggest that the KMT-2 cells are myelomonocytic cells, probably of immature progenitor origin. Besides IL-3, granulocyte-macrophage colony-stimulating factor supported the growth of the KMT-2 cells, but IL-1 alpha, IL-2, IL-4, IL-5, and erythropoietin did not. IL-6 showed only slight activity. Binding studies with 125I-labeled recombinant human (rh) IL-3 indicated that IL-3 bound to a single class of high affinity receptors (approximately 4,000 receptors/cell) on KMT-2 cells with a kd of approximately 200 pmol/L. The chemical cross-linking assay demonstrated that radiolabeled hIL-3 bound three molecules with molecular masses of 170, 130, and 70 Kd. Present data suggest that the newly established human cell line will be a valuable tool for the biologic assay of hIL-3, and a model for biochemical studies of IL-3 receptors.

Eight patients suffering from papulovesicular light eruption (PVLE) were phototested for reproduction of skin lesions with FL20SE and FL20BLB. Of the 8 patients, 50.0% developed typical lesions of PVLE at the test site by provocative phototesting with FL20SE (3 daily exposure of 2MED). In these patients, similar lesions were induced by the provocative test with FL20BLB for 2 consecutive days without filter, but no eruptions were induced when a sharp cut filter UV-35 was used. In conclusion, it is important to consider not only UVB but also the border spectrum between UVB and UVA (320-340 nm) as the action spectrum of PVLE.

Thirteen patients with metastatic liver carcinoma were treated with hepatic irradiation, leukopheresis and monoclonal antibodies to test the tumoricidal effects of murine 17-1A immunoglobulin G (IgG) Mab. A significant increase in survival for this group of patients was identified.

We have identified an endothelin (ET) binding protein on the membranes of human placenta and purified it to homogeneity. It is a polypeptide with an apparent Mol. Wt. of 40,000 and is a major protein to be labeled by cross-linking with either 125I-ET-1, -2, or -3. Binding studies with Scatchard analysis indicated the presence of a single class, high-affinity binding site with Kds of 57 pM, 480 pM and 40 nM for 125I-labeled ET-1, ET-2 and ET-3, respectively. These results suggest that the 40K protein is a major ET receptor in placenta and, most likely, can bind differentially to ET-1, ET-2 and ET-3.

The 5' and 3' ends of the lux mRNA of Vibrio harveyi, which extends over 8 kilobases, have been mapped, and two new genes, luxG and luxH, were identified at the 3' end of the lux operon. Both S1 nuclease and primer extension mapping demonstrated that the start site for the lux mRNA was 26 bases before the initiation codon of the first gene, luxC. The promoter region contained a typical -10 but not a recognizable -35 consensus sequence. By using S1 nuclease mapping the mRNA was found to be induced in a cell density- and arginine-dependent manner. The DNA downstream of the five known V. harveyi lux genes, luxCDABE, was sequenced and found to contain coding regions for two new genes, designated luxG and luxH, followed by a classical rho-independent termination signal for RNA polymerase. luxG codes for a protein of 233 amino acids with a molecular weight of 26,108, and luxH codes for a protein of 230 amino acids with a molecular weight of 25,326. The termination signal is active in vivo as demonstrated by 3' S1 nuclease mapping, confirming that the two genes are part of the V. harveyi lux operon. Comparison of the luxG amino acid sequence with coding regions immediately downstream from luxE in other luminescent bacteria has demonstrated that this gene may be a common component of the luminescent systems in different marine bacteria.

We have purified nascent DNA molecules from Escherichia coli pulse-labeled with 5-bromo[6-3H]deoxyuridine by repeated chromatography on nitrocellulose and isopycnic centrifugation in CsCl. The nascent molecules were labeled with 32P either at their 5' ends using polynucleotide kinase or at their 3' ends using terminal transferase. Compared to the non-nascent DNA of normal density, the nascent dense DNA contained a higher proportion of molecules terminated at their 5' ends with ribonucleotides. Exposure of the dense DNA to alkali generated 5' OH termini quantitatively equivalent to the number of molecules bearing 5' ribonucleotides. Experiments designed (1) to detect structures at the 5' ends of phosphatase-treated nascent DNA molecules that caused them to be resistant to hydrolysis by spleen exonuclease or (2) to detect polypeptides that were associated covalently with small DNA molecules and could be iodinated with the Bolton-Hunter reagent did not yield positive results. We conclude that many, if not all, of the intermediates in E. coli DNA replication are initiated with one or more ribonucleotides. The nascent molecules are outnumbered by small non-nascent DNA molecules in the cell, many of which appear to become slightly longer when cells are pulsed with thymidine. Many of the non-nascent DNA molecules behave as if they were self-complementary or crosslinked.

An eleventh case of heavy (Hgamma1) chain disease (Yok), surviving for more than 10 years and still living showed clinical and pathological findings similar to cases described in the past. The patient was given only glucocorticosteroids, ACTH, antibiotics and gamma globulin, as specific drugs. Precipitation arcs besides the major ones formed by albumin and Fc fragment were disclosed by immunoelectrophoresis. The existence of these minor components were confirmed with antigen-antibody crossed electrophoresis and Sephadex G-200 gel filtration. They did not form precipitation arcs with the other antigens available and they appeared in the same fractions of IgG on gel filtration suggesting their having higher molecular weight than the major ones. In addition to these findings, the clinical course of the patient is described.

Latex particles were covalently linked to the 5'-proximal region of oligo(dT)30. The resultant oligo(dT)30-Latex was tested for its hybridizability to poly(A) containing mRNA. Several advantages were noted as compared to the conventional oligo(dT)30-cellulose column chromatography; (1) a highly efficient (approximately 95%) hybridization occurs in a short reaction period (10min), (2) more than 95% of poly(A) mRNA can be recovered from oligo(dT)30-Latex by a simple heating followed by brief centrifugation, (3) multiple samples can be handled simultaneously and moreover, (4) the poly(A)-mRNA on the oligo(dT)30-Latex can be directly transcribed by AMV reverse transcriptase to form the cDNA. These properties of oligo(dT)30-Latex promise an excellent reagent for nucleic acid technology.

An abnormal protein with similar antigenic properties to Fc fragments of IgG, was found in the serum and urine of an eleventh case of heavy (Hgamma1) chain disease (Yok). This protein was purified with ammonium sulfate precipitation and by column chromatography of DEAE cellulose, CM cellulose and Sephadex G-200. The purity of the protein obtained was 98.5%. It was crystallized easily, forming thin hexagonal plates of various sizes. The chemical compositions and physical properties of the protein including viscosity, partial specific volume, diffusion constant, sedimentation constant, frictional ratio, extinction coefficient and iso-ionic point are reported.

Thirty patients suffering from polymorphous light eruption (PLE), selected by criteria pointing to UVB sensitivity, were phototested for reproduction of skin lesions. Twenty-seven patients (90.0%) had symptoms compatible with or very similar to papulovesicular light eruption (PVLE). Of the 30 patients, 56.7% developed typical lesion of PLE at the test site by provocative phototesting with UVB. Eruptions of the immediate onset type were apt to be reproduced by multiple repeated irradiation, but those of the delayed onset type were reproduced by a single high-dose exposure. In addition, pruritus at the test site seen in all patients was thought to be an important diagnostic symptom. It was obvious that UVB played an etiological role in PLE, including PVLE.

In vitro quantitative biosynthetic studies were carried out on bone marrow cells obtained from an eleventh case with gamma heavy chain disease. The findings indicate that neither cytoplasmic nor extracellular degradation was responsible for the presence of the gamma heavy chain fragment in serum. The absence of a covalent-bound light chain was also confirmed.

A cDNA fragment coding for human c-myc was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Insect cells infected with the recombinant virus produced significant amounts of c-myc protein, which constituted the major phosphoprotein component in these cells. By immunoprecipitation and immunoblot analysis, two proteins of 61 and 64 kilodaltons were detected with c-myc-specific antisera. The insect-derived proteins were compared with recombinant human c-myc-encoded proteins synthesized in Escherichia coli and Saccharomyces cerevisiae cells. The c-myc gene product was found predominantly in the nucleus by subcellular fractionation of infected insect cells.

In vitro stimulation of human lymphocytes were studied in connection with cell fusion. When splenic lymphocytes were stimulated with human cytomegalovirus (CMV), they produced IgG but not IgM antibody against CMV. The stimulation with 50 ng/ml of CMV antigen induced the maximum antibody response, and higher concentrations of CMV antigen decreased antibody response and increased nonspecific IgG production. Human splenic lymphocytes were stimulated for 6 days with CMV antigen (50 ng/ml) and/or B-cell growth factor (BCGF), and then fused with mouse myeloma cells. Stimulation with a combination of antigen and BCGF were able to generate CMV-specific hybridomas synergistically. Two of these hybridomas were cloned by limiting dilution. The human monoclonal antibodies produced by them, C1 and C23, bound to CMV but not to other herpesviruses. C23 neutralized virus infectivity C1 did not at all. This method for generation of hybridomas producing human monoclonal antibodies against a predefined antigen may be applicable to a variety of viral antigens.

Three sex-transforming genes (ix, tra-D, and dsx) in D. melanogaster were examined with respect to possible interactions with the NSR strain SR-spirochetes. The SR-spirochetes exerted a lethal effect on XY but not on XX individuals, regardless of whether they were phenotypically males, intersexes, or females. These results, taken together with those reported by Sakaguchi and Poulson (1963)on tra and 2X3A intersexes, both of which are resistant to the androcidal action of SR-spirochetes, support the interpretation that male susceptibility is a consequence of the single X condition.

The influence of untranslated 5' sequences on c-myc expression was compared by measuring the translational efficiencies of mRNAs which contain leaders derived from exon 1 or intron 1 of the human c-myc gene. Expression plasmids were constructed and introduced into COS cells, and the levels of c-myc mRNA and protein were examined. Our results show that mRNAs transcribed from constructs containing exon 1 or intron 1, which have different folding potential, are translated with approximately equal efficiencies. This suggests that the translation of c-myc mRNA is not controlled by secondary structure alone. In addition, we observed that transcripts in which exon 1 was deleted are not translated more efficiently, but are present at a higher steady-state level. Thus, this example provides evidence for possible control at the transcriptional level. Finally, since the c-myc product was produced in each of our test systems, the results suggest that this protein does not regulate its own transcription or translation via a specific interaction with c-myc exon 1 alone.

When isogenic strains of Escherichia coli, RR1 (rec+) and HB101 (recA), were transformed with mapped recombinant plasmids known to contain Vibrio harveyi luciferase genes and large regions of DNA flanking on both sides, a small percentage (0.005%) of the colonies expressed high levels of luminescence (up to 10(12) quanta s-1 ml-1) in the absence of added aldehyde. The altered ability to express light was found to be due to a mutation in the host and not to an alteration in the recombinant DNA. When these bright colonies were cured of plasmid, they could be retransformed with cloned V. harveyi gene fragments in cis and in trans to yield luminescent colonies at 100% frequency. The maximum length of V. harveyi DNA required to produce light-emitting E. coli was shorter (6.3 kilobase pairs) than that required for expression of the V. fischeri system in E. coli. Cell extracts from bright clones contained wild-type levels of activity for the heteropolymeric (alpha beta) luciferase; fatty acid labeling revealed the presence of the three acylated polypeptides of the fatty acid reductase system which is involved in aldehyde biosynthesis for the luminescence reaction. The increased light emission in the mutant bacteria appeared to arise in part from production of higher levels of polycistronic mRNAs coding for luciferase.

We investigated the bioactivities of the recombinant DNA-derived methionyl 20K hGH (20K-Met-hGH) and methionyl hGH (22K-Met-hGH). The growth-promoting activities in Nb2 cells of 20K-Met-hGH and 22K-Met-hGH were 10.7% and 93.5% of pituitary hGH (P-hGH), respectively. In the IM-9 lymphocyte assay, the binding activities of 20K-Met-hGH and 22K-Met-hGH to hGH receptor were 29.0% and 87.1% of P-hGH, respectively. Our data demonstrate that 20K-Met-hGH may have weaker biological potency than P-hGH.