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A 71-year-old Japanese woman presented with erythematous plaques on the eyelids and subcutaneous indurations or nodules with or without overlying erythema on the hands, thigh, and leg. She also had oral ulcers, arthralgia and a low grade fever. Laboratory tests revealed an elevated titer of antinuclear antibody, an increased erythrocyte sedimentation rate and anemia. Skin biopsy specimens from the hand and thigh showed lymphocytic perivascular and periappendageal infiltrates and vacuolar alterations at the basement membrane zone of the skin appendages. Moreover, there was a dense lymphocytic infiltrate deep in the dermis with extension into the subcutaneous fat, which was compatible with the diagnosis of lupus erythematosus profundus. Although the biopsy specimen from the eyelid lesion did not contain the subcutaneous fat, the changes in the dermis were essentially the same as those of the hand and thigh. The eruption as well as the other symptoms promptly responded to oral prednisolone.

p27Kip1, a cyclin-dependent kinase inhibitor, is a negative regulator of the cell cycle, and apoptosis is a genetically encoded program of cell death. To clarify the relationship between the cell cycle and apoptosis, we investigated expression of p27, cyclin D1 and apoptosis-related proteins (p53, Bax, Bcl-2 and c-Myc) in 60 cases of oral and oropharyngeal squamous-cell carcinoma (SCC) using an immuno-histochemical approach, and evaluated spontaneous apoptosis in vivo. Our most notable finding was that spontaneous apoptosis in the p27-positive group was significantly higher than that in the p27-negative group (p = 0.028). In addition, the percentage of p27-positive cells was clearly correlated with that of Bax-positive cells (gamma = 0.288, p = 0.028) and with that of cyclin D1-positive cells (gamma = 0.416, p = 0.002). Expression of p27 was inversely associated with the clinical stage of total tumor progression (p = 0.027). However, no correlation was found between p27 expression and the following parameters: gender, tumor size, lymph node metastasis, overall survival and disease-free survival. Our results give evidence that the action of the cell-cycle regulator p27 is closely linked with apoptosis in clinical samples from patients and indicate that over-expression of p27 might induce apoptosis in cancer cells through elevation of Bax expression, thereby acting on tumor progression.

Gastrin-releasing peptide (GRP) is present in the lung and functions as a growth factor for bronchial epithelial cells and fibroblasts. GRP may stimulate release of cytokines from alveolar macrophages. However, in interstitial lung diseases, the role of GRP has not been clarified, in part because of the instability of GRP. Progastrin-releasing peptide (ProGRP) molecules are the actual GRP gene products. ProGRP molecules contain common extension peptides(31-98) [ProGRP(31-98)], which are not homologous with other proteins unlike GRP. With the ELISA, we measured ProGRP(31-98) concentrations in sera and bronchoalveolar lavage (BAL) fluids from patients with sarcoidosis and idiopathic pulmonary fibrosis (IPF). Significant increased ProGRP(31-98) concentrations were found in sera and BAL fluids from patients with IPF or sarcoidosis when compared with healthy subjects. Serum ProGRP(31-98) values significantly correlated with BAL fluid ProGRP(31-98) values. In IPF and sarcoidosis, the release of the actual GRP gene products is increased in the lung and the bloodstream, and GRP may play a role during the processes of inflammation and remodeling in interstitial lung diseases.

Fructose-1,6-bisphosphatase deficiency is an inheritable disorder of gluconeogenesis. Sequence analysis of the cDNA of the fructose-1,6-bisphosphatase mRNA isolated from monocytes from a girl with this disease and her consanguineous parents revealed that the patient and her parents were a homozygote and heterozygotes for an insertion of one G residue at G957GGGG961, respectively. This mutation resulted in translation of a truncated enzyme protein, and the mutant protein showed no fructose-1,6- bisphosphatase activity in an overexpression experiment in Escherichia coli. However, this mutation is located in a region of the amino acid sequence which is not well conserved among mammals. A mutagenized clone was prepared from the normal clone. The extents of substitutions and deletions of the amino acid sequence were predicted to be less in the mutagenized protein than in the mutant protein. This mutagenized clone also expressed no fructose-1,6-bisphosphatase activity, although both of two normal clones from control monocytes and a control liver sample expressed an apparently normal level of fructose-1,6-bisphosphatase activity. Thus, this mutation is concluded to be responsible for fructose-1,6-bisphosphatase deficiency in this patient.

We investigated phenotypic surface markers of peripheral blood lymphocytes including expression of γδ T cell receptor (TCRγδ) in 185 patients with sarcoidosis and 42 normal subjects. The pr

Congenital self-healing reticulohistiocytosis is a variant of Langerhans cell histiocytosis. It is present at birth or appears in the neonatal period and involutes spontaneously within 3 to 4 months. Although the skin eruptions in most cases consist of papulonodules, several patients with vesicular or bullous lesions have been reported. We describe a case of congenital self-healing reticulohistiocytosis presenting hemorrhagic bullae that mimicked hemangioma. (J Am Acad Dermatol 2003;48:S75-7.)

We measured eosinophilic cationic protein (ECP) concentrations in the circulation and bronchoalveolar lavage (BAL) fluids from patients with chronic eosinophilic pneumonia, patients with eosinophilic granuloma, and normal control subjects. Significantly increased ECP concentrations were found in the circulation of patients with chronic eosinophilic pneumonia and with eosinophilic granuloma compared with those found in control subjects. The ECP concentrations were well correlated to eosinophil counts in the circulation of patients with chronic eosinophilic pneumonia, while they were not in patients with eosinophilic granuloma. Chronic eosinophilic pneumonia patients had prominently increased ECP concentrations in BAL fluids compared with those found in control subjects, while eosinophilic granuloma patients did not. Those concentrations in chronic eosinophilic pneumonia patients were well correlated to eosinophil counts in the BAL fluid. Corticosteroid therapy remarkably decreased circulating ECP concentrations in three patients with chronic eosinophilic pneumonia, but it had no significant effects in two patients with eosinophilic granuloma. Measurement of ECP concentrations seems to be useful to evaluate the disease activity of chronic eosinophilic pneumonia.

Polyomavirus enhancer binding protein, PEBP2 (PEA2), is a heterodimer of two distinct subunits, alpha and beta, of which the former directly binds to DNA and the latter acts auxiliary to enhance the DNA binding. Recent cloning studies has revealed that the alpha subunit is homologous to the products of the Drosophila segmentation gene runt and the human AML1 gene, and that it functions as a major regulator for the T cell-specific gene expression. We have currently cloned cDNAs for the beta subunit. The isolated cDNAs contain three isoforms that are presumed to arise from alternative RNA splicing and encode polypeptides consisting of 187, 182, and 155 amino acids, respectively. These polypeptides neither show any significant homology with known other proteins including the alpha subunit nor have any known DNA-binding and dimerization domains. Thus, PEBP2, as the complex of these subunits, is thought to constitute an entirely novel category of heteromeric transcriptional regulator together with the Runt and AML1 proteins. Gel retardation assays of the cDNA-encoded proteins produced in an in vitro translation system or in Escherichia coli demonstrated that the larger two beta isoforms, but not the smallest one, can dimerize with the alpha subunit. Furthermore, this heterodimerization was shown to cause a marked increase in the intrinsic DNA binding affinity of the alpha subunit.

Background and Methods. The prevalence of type 6, 11, 16, 18, and 33 human papillomavirus (HPV) was investigated with the polymerase chain reaction (PCR) on formalin-fixed, paraffin wax-embedded material, including 48 neoplastic and 21 normal urinary bladder specimens. The PCR-amplified DNA were analyzed by gel electrophoresis and dot blot and Southern blot hybridization. Some tissues were tested further by nonisotopic in situ hybridization. Results. HPV DNA was detected in 39 (81%) of 48 carcinomas and 7 (33%) of 21 normal urinary bladder specimens, The presence of high-risk HPV (types 16,18, and 33) was increased significantly in carcinoma cases (62%) as compared with normal specimens (14%) (P < 0.01). Similarly, multiple HPV infections were significantly higher in carcinoma (60%) than in the normal tissues (5%) (P < 0.01). The overall and high-risk HPV infections in both neoplastic and normal specimens were distributed almost equally in male and female patients. There was no significant correlation between positive results for HPV and histologic grades of the carcinoma. Conclusions. These results demonstrate that the urinary bladder in both sexes is another site where infection with the common genital tract HPV may carry a risk of malignant transformation. Cancer 1992; 70:1967-1973.

The core sequence of the enhancer of murine leukemia virus (MuLV) long terminal repeat is highly conserved in a large number of MuLV strains and appears to play an essential role when SL3-3 or Moloney strains induce T cell lymphoma in mice. We found by using the electrophoretic mobility shift assay that a polyomavirus enhancer core-binding protein, PEBP2, bound to this core motif of MuLV. We also noted that PEBP2 in several hematopoietic cell lines derived from B lymphocyte, macrophage and myelocyte lineages migrated significantly faster than the authentic PEBP2 detected in NIH3T3 (ibroblasts. Interestingly, PEBP2 detected in the cell lines of T lymphocyte lineage appeared to contain both types, which were indistinguishable in electrophoretic mobility from those of NIH3T3 and of B lymphocyte, macrophage and myelocyte lineages. The treatment of the nuclear extract containing PEBP2 with phosphatase generated PEBP3, which is a subcomponent of PEBP2 and retained the same DNA-binding specificity as PEBP2. The altered mobility of hematopoietic cell-derived or T lymphocyte-derived PEBP2 was found to be due to the alteration of the mobility of PEBP3. Based on the distinct mobility of PEBP2/3 of T lymphocytes from those of other hematopoietic cells, we discuss the implication of PEBP2 in MuLV-induced T cell leukemia and T cell-specific gene expression.

Survivin, a new member of the inhibitor of apoptosis protein (IAP) family, has been reported to be expressed in many cancers but not in differentiated normal tissues. In the present study, we describe the identification of a novel alternatively spliced survivin transcript, designated as survivin-3B. It comprises 5 exons including novel exon 3B derived from a 165-bp long portion of intron 3. Acquisition of a new in-frame TGA stop codon within the novel exon 3B results in an open reading frame (ORF) of 363 nucleotides, predicting a truncated 120 amino acid protein. Expression of survivin-3B was detected in human colon and gastric adenocarcinoma cell lines as well as mononuclear cells prepared from patients with myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). Survivin-3B contains a single baculovirus IAP repeat (BIR), which is critical for apoptosis inhibition. However, it lacks a carboxyl-terminal coiled-coil domain, suggesting that survivin-3B may not be associated with G2/M phase. These data indicate that the function of survivin-3B may be different from that of regular survivin.

Survivin, a unique member of the inhibitor of the apoptosis protein (IAPs) family, is over-expressed in many cancers but not in normal differentiated adult tissues. Using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we investigated patterns of survivin gene expression in a group of 12 patients with chronic myeloid leukemia (CML) representing both chronic and blastic phases of the disease. All 6 patients in chronic phase CML were uniformly negative for the survivin transcript, in contrast to 4 Philadelphia chromosome-positive (Ph+) CML patients in blastic crisis, all of whom (100%) were positive for survivin with tangible levels of expression. However, survivin expression was markedly down-regulated in 2 atypical CML patients with Philadelphia chromosome-negative (Ph-) blastic crisis. Our data indicates that up-regulation of survivin expression may be involved in typical CML evolution from the chronic into the blastic phase of the disease.

International audience; We previously isolated a 34-kDa nuclease (AN34) from apoptotic human leukemia cells. Here, we identify AN34 as an N-terminally truncated form of human AP endonuclease (Ape1) lacking residues 1-35 (delta35-Ape1). Although Ape1 has hitherto been considered specific for damaged DNA (specific to AP site), recombinant AN34 (delta35-Ape1) possesses significant endonuclease activity on undamaged (normal) DNA and in chromatin. AN34 also displays enhanced 3'-5' exonuclease activity. Caspase-3 activates AN34 in a cell-free system, although caspase-3 cannot cleave Ape1 directly in vitro. We also found that Ape1 itself preferentially cleaves damaged chromatin DNA isolated from cells treated with apoptotic stimuli and that silencing of Ape1 expression decreases apoptotic DNA fragmentation in DFF40/CAD-deficient cells. Thus, we propose that AN34 and Ape1 participate in the process of chromatin fragmentation during apoptosis.

Survivin is a member of the inhibitor of apoptosis protein (IAPs) family and considered to play a pivotal role in oncogenesis. We present the first report of survivin expression profile in myelodysplastic syndrome (MDS). Expression of survivin messenger RNA was evaluated by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in patients with MDS and acute myeloid leukemia (AML). Eleven out of 12 patients with refractory anemia (RA) (91.6%), and all 3 patients with refractory anemia with excess blasts in transformation (RAEBt) (100%), were positive for survivin expression with the majority of cases showing abundant levels of the survivin transcript. On the other hand, expression of survivin was undetectable in the 4 patients with chronic myelomonocytic leukemia (CMMoL). The level and frequency of survivin expression in patients with refractory anemia were compared to those in patients with AML. Out of 12 patients with de novo AML, 5 patients (41.7%) showed detectable levels of survivin expression. Abundant survivin expression in RA was also confirmed by immunohistochemistry. In contrast, survivin was almost absent in two cases with aplastic anemia. We propose that high levels of survivin expression can serve as a reliable diagnostic marker of RA in MDS.

Fructose-1,6-bisphosphatase (FBPase) is a key gluconeogenic enzyme. The data herein show that both the enzyme activity and mRNA level of the human FBPase gene are enhanced by 9-cis retinoic acid (9cRA) and all-trans retinoic acid (atRA) as well as by 1,25-dihydroxyvitamin D3 (VD3) in human promyelocytic HL60 cells and normal monocytes in peripheral blood, which were used as an alternative source to liver for the DNA diagnosis of FBPase deficiency. To understand the molecular mechanism of this enhancing action, the 2.4 kb 5'-regulatory region of the human FBPase gene was isolated and sequenced. Using luciferase reporter gene assays, a 0.5 kb FBPase basal promoter fragment was found to confer induction by VD3, 9cRA, and atRA that was mediated by the vitamin D3 receptor (VDR), retinoid X receptor (RXR), and retinoic acid receptor (RAR). Within this region, a direct repeat sequence, 5'-TAACCTttcTGAACT-3' (-340 to -326), which functions as a common response element for VD3, 9cRA, and atRA, was identified. The results of electrophoretic mobility shift assays indicated that VDR-RXR and RAR-RXR heterodimers bind this response element. Collectively, these observations indicate that VD3 and RA are important modulators of the expression of the human FBPase gene in monocytic cells.

A 53-year-old female with a 9-month history of chest pain presented with a giant anterior chest wall mass. Radiologic examination showed an anterior mediastinal tumor invading the chest wall. Serum calcium and parathyroid hormone-related protein (PTHrP) levels were extremely elevated. Biopsy specimens disclosed a squamous cell carcinoma with Hassall’s corpuscle-like keratotic pearls, and an immunohistological study showed a positive staining with PTHrP. The tumor and serum PTHrP concentration markedly decreased following cisplatin-based chemotherapy and radiation. This is the first case of PTHrP producing a thymic carcinoma with the unusual presentation of a large extrathoracic mass.

pMRA17 cloned from Pseudomonas K-62 plasmid pMR26 specified the resistance to both organic and inorganic mercurials. DNA sequence of this broad-spectrum resistant mer operon was determined. The 5504-bp sequence includes six open reading frames (ORFs), five of which were identified as merR , merT , merP , merA and merB in order by analysis of deletion mutants and by comparison with the DNA and amino acid (aa) sequences of previously sequenced mer operons. The merB encoding organomercurial lyase showed a less identity than the other mer genes with those from other broad-spectrum resistance operons. The remaining ORF named merE , located between merA and merB , had no significant homology with the published mer genes and seemed to be a new gene which may involve in phenylmercury resistance. Induction experiments and maxicell analyses of the mer -polypeptides revealed that pMRA17 mer operon expressed mercurial-inducible phenotype and the merB and merE as well as the merA were under the control of MerR which could activate not only by mercuric ion but also by organomercurials. © 1997 Elsevier Science B.V. All rights reserved.

Fructose 1,6-bisphosphatase deficiency is an autosomal recessive inherited disorder of gluconeogenesis. We could isolate cDNAs encoding human fructose 1,6-bisphosphatase from normal monocytes, liver and kidney, but not from normal lymphocytes. The cDNAs contained an open reading frame coding for 338 amino acids, and their nucleotide sequences in monocytes and liver were identical. G644C645 nucleotides in this sequence were the same as those of cDNA from HL-60 cells, although our result differed from a previous report (M. El-Maghrabi et al. (1993) J. Biol. Chem. 268, 9466-9472) on an alteration to C644G645 nucleotides in human liver cDNA resulting in a change of Gly-214 to Ala-214 in the enzyme. The Gly-214 (GGC) residue was therefore conserved in the enzymes hitherto isolated from humans and other animals. Analysis of monocytes in seven patients with fructose 1,6-bisphosphatase deficiency showed a DNA fragment with apparent normal size in two sisters but no detectable DNA fragment in the other five patients. Monocytes were thus useful as an alternative source for mRNA from human liver for the molecular analysis of fructose 1,6-bisphosphatase deficiency.

cDNAs representing the alpha subunit of polyomavirus enhancer binding protein 2 (PEBP2; also called PEA2) were isolated. The products of the cDNAs are highly homologous to that of Drosophila segmentation gene runt (run) for an N-proximal 128-amino acid region showing 66% identity. The run homology region encompasses the domain capable of binding to a specific nucleotide sequence motif and of dimerizing with the companion beta subunit. The human AML1 gene related to t(8;21) acute myeloid leukemia also had a run homology region. Together with the beta subunit, which increases the affinity of the alpha subunit to DNA without binding to DNA by itself, PEBP2 represents a newly discovered family of transcription factor. The major species of PEBP2 alpha mRNA was expressed in T-cell lines but not in B-cell lines tested. Evidence indicated that PEBP2 functions as a transcriptional activator and is involved in regulation of T-cell-specific gene expression.

We examined the expression of tumor-suppressor protein p53 in a variety of laryngeal carcinomas from 43 patients (25 primary, 13 metastatic and 5 recurrent cases), 13 normal laryngeal tissues and 7 benign laryngeal nodule biopsy specimens, using the polyclonal antibody CM-1. Previously we have reported a high incidence of ras mutations (51%) and human papillomavirus (HPV) infection (37%) in these laryngeal carcinomas. p53 protein was detected by immunohistochemistry in 65% of laryngeal carcinomas (60% of primary, 69% of metastatic and 80% of recurrent cases). No correlation was found between p53 over-expression and histological grading of the tumors. None of the specimens from normal larynx and laryngeal nodules revealed any detectable level of this protein. Furthermore, 11 (69%) of 16 HPV-positive cases and 17 (77%) of 22 cases with ras mutation showed variable grades of p53 expression. Twelve (71%) of 17 laryngeal carcinomas in current study having both p53 over-expression and ras mutation were moderately or poorly differentiated. Likewise, positivity for these 2 parameters was significantly increased in metastatic tumors (9 of 13 cases, 69%) as compared with primary and recurrent tumors (8 of 30 cases, 27%) (p < 0.01). Our results suggest that multiple factors are involved in this malignancy, and that the simultaneous over-expression of p53 and the presence of ras mutation may be related to the progression stage of laryngeal carcinoma.

In order to determine the prevalence and type-specific distribution of human papillomavirus (HPV) in women from Pakistan, a country with a low cervical cancer rate (hospital-based data), and to compare these results with their counter-parts in Japan, we studied 56 non-neoplastic cervical tissues and 162 cervical carcinoma cases from both countries. HPV infection was defined by in situ hybridization in paraffin-embedded tissues using biotinylated HPV 6/11, 16 and 18 probes. The overall positivity rates in non-neoplastic and neoplastic cervical tissues from Pakistan were 33% and 88%, while in those from Japan the rates were 46% and 80%, respectively. High-risk HPVs (16 & 18) were found in 17% of the non-neoplastic specimens and in 69% of cervical carcinoma cases from Pakistan, while Japanese figures in this respect were 19% and 68%, respectively. No correlation was found between the type-specific distribution or prevalence of HPV and the geographic location of the cases examined in the 2 countries. However, in comparison to Japanese women, the incidence of HPV-16-positive cervical carcinoma in Pakistani women decreased significantly (p less than 0.05) in the oldest age group as compared to the youngest age group.

Growth of Proteus mirabilis harboring R100-1 ( fi + drd str r cml r tet r sul r ) factors in Penassay broth containing sodium dodecyl sulfate (SDS) leads to the loss of all or part of the genetic elements in high frequencies. In media containing SDS at concentrations as low as 0.03%, both lysis of R + cells and elimination of the R factors occur at high frequencies. Appearance of drug-susceptible cells in R + cultures occurs during the exponential phase of growth; however, the frequencies of susceptible cells increase substantially after the culture reaches the stationary phase. Reconstruction experiments, coupled with other observations, suggest that the major factor in altering the frequency of drug-susceptible variants is the greater resistance of the variants to the lytic action of SDS. This resistance correlates in most cases with the loss of the transfer functions in the resistance transfer factor.

DNA replication of plasmid R6K initiates at three unique sites, ori alpha, ori beta, and ori gamma. Replicating DNA molecules of a deletion derivative of R6K were synthesized in an in vitro system containing pi protein fraction from cells carrying a mini-R6K derivative that produced only this initiation protein as an R6K-encoded protein and analyzed by electron miscroscopy. Requirement of pi protein for the activity of all these three replication origins in vitro was verified. Frequencies of initiation at the three origins were almost equal.

A soluble extract prepared from cells of an Escherichia coli strain carrying the antibiotic resistance plasmid R6K is capable of carrying out the complete process of R6K DNA replication. DNA synthesis in vitro is dependent on the four deoxyribo- and ribonucleotide triphosphates and is sensitive to rifampin and streptolydigin, inhibitors of DNA-dependent RNA polymerase. The incorporation of deoxyribonucleotides into R6K DNA also is sensitive to actinomycin D, novobiocin, arabinofuranosyl-CTP, and N-ethylmaleimide. Kinetics of synthesis are linear for 60 to 120 min. Replication proceeds semiconservatively and supercoiled closed-circular DNA molecules are synthesized. Analysis by alkaline sucrose gradient centrifugation indicated that the early R6K DNA products contain DNA fragments of approximately 18 S in size, corresponding to the length between the R6K alpha origin of replication and the terminus of replication observed in vivo. Addition of exogenous supercoiled R6K DNA is inhibitory to the in vitro system, whereas the addition of R6K DNA in the form of relaxation complex stimulates R6K DNA synthesis to a small extent.

A non-self-replicating segment (1370 base pairs) of plasmid R6K was cloned in E. coli and shown to trans-complement temperature-sensitive replication mutants of this plasmid. This segment contains the gene which codes for a protein required for initiation of replication of the plasmid, and was used as a helper in a functional assay for an origin of replication in R6K derivatives. A 420 bp fragment, derived from R6K DNA, was shown to carry a functional origin since it was capable of replicating as a plasmid in E. coli cells carrying the helper segment either on the host chromosome or on a plasmid Col E1 derivative. The copy number of the origin fragment in cells carrying the helper segment on the chromosome is essentially the same as the copy number of R6K. A model for the positive regulation of plasmid R6K replication is presented.

Glycine, known to inhibit the synthesis of a peptidoglycan component of the bacterial cell wall, was effective in eliminating drug resistance of Escherichia coli K12 JE2100 strain harboring the R100–1 factor, although in lower frequencies than that of sodium dodecyl sulfate (SDS). The action of glycine was found to be less effective on the same R factor in JE177 strain, and not effective on the F factor in W6. Infection of R factors from R+ cells to R– cells was found to take place in the glycine broth as efficiently as in broth without glycine. This might result in lowering the apparent efficiency of the action of glycine on those plasmids. The segregation patterns of drug-susceptible clones obtained by the glycine treatment were different from those obtained after the SDS treatment. These results coupled with other evidences suggest that the mode of action of glycine on R+ cells may be different from those of other curing agents and may involve mechanisms other than selection of R– or drug-susceptible segregants that are present in R+ culture.

A specific action of sodium dodecyl sulfate (SDS) on the sex (F) factor in the integrated state of Escherichia coli K-12 Hfr H strain is reported. Growth of Hfr cells in Penassay Broth containing SDS results in the elimination of part or all of the F factor, yielding low and nonfertile variants of defective Hfr type and F + cells and also F − derivatives. Appearance of such variants was generally observed after the culture reached stationary phase. The frequencies of F − cells then increased. F − cells were usually isolated as the major population among survivors. Some defective variants of Hfr cells with an intermediate fertility between standard Hfr and F + cells had lost sensitivity toward the male-specific ribonucleic acid phage M12. Other defective Hfr variants with as much or less fertility than standard F + cells had also all lost sensitivity to phage M12. On single-colony isolation, they segregated nonfertile female H cells which, when infected with F, could restore high fertility with oriented transfer of the chromosome the same as that of the original Hfr H. Also, sensitivity to phage M12 was regained. Female H cells were characterized as those lacking fertility but still retaining a small segment of F or sfa locus at the original part of the chromosome, where newly infected F could attach. Similar results were obtained with two other Hfr strains. A possible mechanism of the specific action of SDS is discussed.

Evidence is presented for the specific role of sex pili in the eliminatory action of sodium dodecyl sulfate (SDS) on sex (F) and drug resistance (R) factors in Escherichia coli K-12 strains leading to their loss. SDS at 0.03% concentration lysed JE3100 F 8 + (F- gal )/ gal − fla − pil − in Penassay broth after they had grown exponentially and reached maximum growth to the extent that the agent at concentrations higher than 1% did. However, the agent was only effective in eliminating sex factors from JE3100 in high frequencies at concentrations higher than 1%. Increase of osmotic pressure of the culture with SDS at concentrations as low as 0.03 to 0.1% by addition of sucrose led to the substantial increase of elimination efficiency. Reconstruction experiments between F 8 + and F − cells in the SDS culture revealed the selective growth of F − cells as well as a delay of maximum growth of F − variants derived from F 8 + cells, compared with those of F 8 + cells, as well as F − cells originally added to the culture. The agent was not very effective in eliminating sex factors from JE3427 F 8 m + 5/ fla − pil − cells which lack the function of production of F pili. F 8 m + 5 cells showed a sensitivity toward SDS intermediate between those of F 8 + and F − cells. SDS was further effective in eliminating R factors from KE132 R 100-1 + / fla − pil − cells in high efficiency; however, the action was not efficient with KE133 F 100 + cells possibly with fewer sex pili than R 100-1 + . Action of acridine orange on these F + or R + strains was found to be different in some aspects from that of SDS.

The efficient catalytic action of polyphosphoric acid, when used in the presence of suitable nucleophiles, of both normal and abnormal ring opening of 4β,5-epoxy-5β-cholestan-3-one (I) is reported. In acetic acid, I affords 2α-acetoxycholest-4-en-3-one (XIIa; abnormal product) whilst, in marked contrast, ethanethiol reacts with I in dioxane affording 4-thylthiocholest-4-en-3-one (XV; normal product) and a further product, 3,4-bis-(ethylthio)cholesta-3,5-diene (XVI). Ethanedithiol and β-mercaptoethanol react with I, as expected, affording cholesta-3,5-dieno[3,4-b]dithiane (XVII) and its oxathiane derivative (XIX) respectively. The nature of the reactions is briefly discussed and presence of some unique conjugation in the SC 3 S and OC 3 C 4 S systems in the dithiane (XVII) and oxathiane (XIX) is suggested on the basis of their UV absorption data.

A doublet centered at τ 6·22 in the NMR spectra of 4-ethylthiocholest-4-en-3-one, 17β-acetoxy-4-ethylthioandrost-4-en-3-one, 16α,17α,-epoxy-4-ethylthiopregn-4-ene-3,20-dione, and 17α-hydroxy- and 17α-acetoxy-4-ethylthiopregn-4-ene-3,20-diones, is observed and is attributed to one half of an AB-type quartet ( J = 14·5 c/s) resulting from the 6-methylene protons. The unusually large downfield shift of the doublet assigned to the C06 equatorial proton is presumed to be due to the deshielding effect of the ethylthio function at C-4. The chemical shift of the 6α-proton in cholest-4-en-3-one and its 4-substituted analogs, Me, OH, OMe, OAc, Cl, Br, SH, SAc, S-S-bis and S-bis, are tabulated and the deshielding effect of alkylthio group is found to be more than 1 ppm which is the srongest among these various functions. The unique deshielding effect of alkylthio function is shown to be general by examination of 2-ethylthio-3,5,5-trimethylcyclohex-2-enone and its 2-substituted analogs. An anomalously weak deshielding effect by the SH group on a double bond is pointed out.

The in vitro effect of 4-nitropyridine 1-oxide (4NPO) and its reductive erivatives on the transforming DNA in Bacillus subtilis was investigated. Among six compounds used, only 4-hydroxylaminopyridine 1-oxide (4HAPO) was effective to inhibit the transforming activity of DNA. The inhibition took place only in the presence of air.This led to a conclusion that 4HAPO itself is not the ultimate compound in the inhibition process of the transforming DNA but an oxidation product possibly of free radical character may be primarily responsible for this event.

Summary Fructose-1, 6-bisphosphatase (FBPase) deficiency is an autosomal recessive inherited disorder and may cause sudden unexpected infant death. We reported the first case of molecular diagnosis of FBPase deficiency, using cultured monocytes as a source for FBPase mRNA. In the present study, we confirmed the presence of the same genetic mutation in this patient by amplifying genomic DNA. Molecular analysis was also performed to diagnose another 12 Japanese patients with FBPase deficiency. Four mutations responsible for FBPase deficiency were identified in 10 patients from 8 unrelated families among a total of 13 patients from 11 unrelated families; no mutation was found in the remaining 3 patients from 3 unrelated families. The identified mutations included the mutation reported earlier, with an insertion of one G residue at base 961 in exon 7 (960/961insG) (10 alleles, including 2 alleles in the Japanese family from our previous report [46% of the 22 mutant alleles]), and three novel mutations—a G→A transition at base 490 in exon 4 (G164S) (3 alleles [14%]), a C→A transversion at base 530 in exon 4 (A177D) (1 allele [4%]), and a G→T transversion at base 88 in exon 1 (E30X) (2 alleles [9%]). FBPase proteins with G164S or A177D mutations were enzymatically inactive when purified from E. coli . Another new mutation, a T→C transition at base 974 in exon 7 (V325A), was found in the same allele with the G164S mutation in one family (one allele) but was not responsible for FBPase deficiency. Our results indicate that the insertion of one G residue at base 961 was associated with a preferential disease-causing alternation in 13 Japanese patients. Our results also indicate accurate carrier detection in eight families (73%) of 11 Japanese patients with FBPase deficiency, in whom mutations in both alleles were identified.

Two kinds of mutations affecting the copy-number control of plasmid R6K were isolated and identified in an initiator π protein by DNA sequencing. Firstly, a temperature-sensitive replication mutation, ts22, with decreased copy number results in a substitution of threonine to isoleucine at position 138 of the 305-amino-acid π protein. Secondly, a high-copy-number (cop21) mutant was isolated from this ts mutant and was identified by an alteration of alanine to serine at position 162. This cop21 mutation suppressed the Ts character and was recessive to the wild-type allele in the copy control.

Possible mechanisms involved in the action of 4-nitropyridine 1-oxide (4NPO) on Escherichia coli K-12 pro + cells in Penassay broth leading to the selective isolation of proA − and/or proB − mutants but not proC − mutant were studied. Reconstruction experiments between pro + and pro − cells, together with experiments on the bactericidal action of 4NPO on pro + and pro − cells, indicated that 4NPO is more toxic for pro + and proC − cells than for proA − and proB − cells. These results, coupled with data indicating little mutagenicity of 4NPO on E. coli cells, led us to conclude that the selection of proA − and/or proB − cells that arose spontaneously in the pro + culture is a possible mechanism for the action of 4NPO. Examination of 4NPO sensitivity of pro + transductants derived from proA − and proB − cells with P1 vir phage and pro + cells as donor and of pro + spontaneous revertants derived from those pro − cells suggested that 4NPO-sensitive gene(s) should be on, or very close to, the proA and proB loci and that both products of proA and proB genes may be involved in the sensitivity of bacteria to 4NPO. The fact that the 4NPO-sensitive allele is dominant over the 4NPO-resistant allele further indicated the possible correlation between gene products of proA and proB and the 4NPO sensitivity of bacteria. Experiments on metabolic conversion of 4NPO with bacterial cells proved that the major metabolic pathway of the agent is reduction to (possibly via 4-nitroso-) 4-hydroxylamino- and 4-amino-pyridine 1-oxides, and then to 4-aminopyridine. Investigation of the effect of structural modification of 4NPO on the elective selection of Pro − mutants in Pro + culture further suggested that the structural feature indispensable for the action of the agent is the hydroxyl-amino or its more oxidized state at the 4 position and the N -oxide moiety at the 1 position on the pyridine skeleton. Action of 4NPO in minimal medium was found to be bacteriostatic on pro + cells but not on pro − cells, leading to the formation of long nonseptate multinucleate filament cells on pro + cells. Possible biochemical mechanisms of the selective toxicity of 4NPO for pro + and pro − cells are discussed.

Regions of the plasmid genome essential for plasmid replication and maintenance in Escherichia coli have been identified for two antibiotic resistance Plasmids, RK2 and R6K. In both cases a small segment of the plasmid, containing an origin of replication, has been maintained as an extrachromosomal element when another essential region(s) of the plasmid that specifies a trans-acting product is present in the host cell. Nucleotide sequence analysis of the origin regions of plasmids RK2 and R6K revealed seven 22 base pair (bp) direct repeats in tandem for the R6K origin region and eight 17 bp direct repeats in clusters of three and five for RK2. A model is presented involving the interacting of the π protein, the trans-acting product of the R6K genome that is required for the initiation of R6K replication, with the 22 bp direct repeats as a necessary step in the initiation of plasmid R6K replication.