Transcriptional gene expression profiles of oesophageal adenocarcinoma and normal oesophageal tissues.

The molecular events associated with the development of adenocarcinoma of the oesophagus are not well understood. Gene expression associated with oesophageal adenocarcinoma was investigated using a cDNA array containing 1,176 human cancer-associated genes. Approximately 59% of the genes were expressed at detectable levels with 15 genes (1.3%) exhibiting differential (> 2.5-fold) expression in either normal oesophagus or adenocarcinoma tissue. Nine genes were up-regulated in oesophageal adenocarcinoma tissue (matrix metalloproteinase 11 (MMP11), ornithine decarboxylase (ODC), cytokeratins 8 and 18, integrin alpha 3 (ITGA3), integrin alpha 6 (ITGA6), BIGH3 (transforming growth factor beta-induced), beta-catenin and CDC25B (M-phase inducer phosphatase 2)). Six genes were down-regulated in adenocarcinoma tissue (cytokeratin 4, plasminogen activator inhibitor 2 (PAI-2), interleukin 1 receptor antagonist (IRAP), cytokeratin 13/15/17, MAD and retinoic acid receptor gamma 1 (RARG)). Many of these differentially expressed genes influence cell-cell adhesion, cell-extracellular matrix and composition, transcriptional activation and cell cycle progression and are likely to contribute to development of oesophageal adenocarcinoma.

Molecular detection and sequencing of "Norwalk-like viruses" in outbreaks and sporadic cases of gastroenteritis in Ireland.

Norwalk-like viruses (NLVs) are now established as the most important causative agents of epidemic gastroenteritis worldwide. The overall objective of this study was to determine the molecular epidemiology of Irish NLV isolates for the first time by obtaining sequence data from specimens originating from outbreaks and sporadic cases of gastroenteritis. Eight samples from sporadic cases of gastroenteritis and nine isolates from separate NLV outbreaks were examined. Of the sporadic isolates, six were shown to be genogroup 2 (G2) by RT-PCR, while two were G1. All of the outbreak isolates were G2. All isolates were partially sequenced within a highly conserved region of ORF1 (RNA-dependent RNA polymerase gene). Sequence data were aligned and a dendogram was constructed. The results indicated that the majority of G2 isolates were seen to cluster with Bristol and Lordsdale virus, while the two G1 specimens were related most closely to Southampton virus. Further downstream sequence analysis of a number of the isolates confirmed this result. It is concluded that the majority of NLV isolates circulating in Ireland belong to the Bristol/Lordsdale clade.

High-resolution detection of loss of heterozygosity of dinucleotide microsatellite markers.

Dinucleotide microsatellite markers are frequently investigated to study inheritance, genetic stability, and allele frequency distribution in a wide variety of genetic disorders. Previous studies have encountered significant problems regarding resolution and detection of dinucleotide, microsatellites. In this study, a useful method to investigate loss of heterozygosity (LOH) of dinucleotide microsatellite markers is described that involves the use of nondenaturing (Spreadex) submerged gel electrophoresis and SYBR Green I nucleic acid staining. This method omits the gel casting step and the use of hazardous radioactive materials frequently used in many microsatellite studies that employ polyacrylamide gel nucleic acid denaturation analysis. Using this method, 62 patients' paired tumor and normal samples were investigated to detect allele deletions in a region of chromosome 7q31.1, which is believed to harbor a tumor suppressor gene. Interpretable results were obtained in all cases. These results were compared to those attained using ABI Prism Genetic Analyzer 310 and Gene-Scan. There were no discrepancies in results obtained between the two assays. The Spreadex system is cheap, does not require larger equipment costs, and may prove to be a useful system for high-throughput investigation of microsatellites. It may have diagnostic significance and also prove useful if applied to population-based genomic screening and linkage analysis.

Rotavirus survival and stability in foods as determined by an optimised plaque assay procedure.

Tissue culture adapted rotavirus strains were propagated in MA104 and CaCo2 cells using standard cell culture procedures. The progress of infection was monitored by examining for a cytopathic effect, and for the presence of viral RNA in the tissue culture supernatant as determined by a guanidinium-based method. Subsequently, an effective plaque assay for rotavirus was developed using MA104 cells by optimising the adsorption time (2 h) and the levels of fetal calf serum (2.5%) in the overlay medium. Tragacanth gum was used in the overlay medium to immobilize the virus, and plaques were subsequently stained with 1% crystal violet. Using this optimised plaque assay, the survival of rotavirus following exposure to heat and UV irradiation was evaluated by enumerating the clear plaques. It was shown that 60 degrees C for 10 min was sufficient to reduce the viral titer by at least 7 logs, and 50 mJ of UV irradiation was sufficient to reduce the initial viral titer by > 2.5 logs. This optimised plaque assay was also used to determine the survival and stability of rotavirus from a range of experimentally contaminated foods including fruit juice, formula milk and lettuce.

Detection of sporadic cases of Norwalk-like virus (NLV) and astrovirus infection in a single Irish hospital from 1996 to 1998.

BACKGROUND: 'Norwalk-like viruses' (NLV) and astroviruses are recognised as the most important etiologic agents of viral gastroenteritis, excluding rotaviruses. However, neither of these two groups of viruses is routinely screened for in Irish hospital laboratories. OBJECTIVE: The objective of this study was to examine faeces collected from patients with non-bacterial, non-rotaviral gastroenteritis and examine if NLVs and astroviruses could be identified as the causative agents of the illness. STUDY DESIGN: Faecal specimens were collected from a single Irish hospital from February 1996 to June 1998. Three hundred and sixty samples were tested for the presence of NLVs using newly designed inosine-containing degenerate primers. Two hundred and three faecal specimens from paediatric patients were screened for the presence of astroviruses. RESULTS: the results of the screening study were that 29 (8%) specimens were found to be positive for NLV by reverse transcription polymerase chain reaction (RT-PCR) and 15 (7%) specimens from paediatric patients were found to be positive for astroviruses. Genotyping of the NLV-positive samples determined that four of the isolates were from genotype I (G1) and 25 were G2. The G2 positive specimens were further subtyped by oligonucleotide probing and the majority (n = 21) were found to be subtype P2-B, with four isolates being typed as P2-A. No P1-B isolates were found. CONCLUSIONS: This is the first report of detection of sporadic cases of NLV and astrovirus in Ireland. The results obtained highlight the need for continued surveillance of these viruses and the development of rapid detection systems for use in clinical laboratories.

Rotavirus gastroenteritis among paediatric patients at Tralee general hospital.

Rotavirus infection among paediatric patients in Tralee general hospital was monitored over 4 years (1994 - 1998). A total of 2,319 specimens from gastroenteritis patients less than 7 years old were tested by latex agglutination assay, of which 203 (8.75%) were deemed positive. An inverse correlation was observed between age and susceptibility to infection, with the very young (under 2 years) most frequently infected. The virus was almost equally distributed among males (53%) and females (47%) testing positive for rotavirus. A distinctive early Spring peak of infection was evident annually, although the largest peak was identified in December 1997. This correlated with a significant change in circulating genotype as determined by RT-PCR based genotyping analysis of 45 rotavirus samples. In 1997, rotavirus accounted for 64% of all identified paediatric enteric agents at Tralee General for which an infectious agent could be identified. The average hospital stay was 3.2 days, and the direct hospital costs for rotavirus associated gastroenteritis was estimated at pounds sterling 31,232 per annum.

Exercise hemodynamic performance of the pulmonary autograft following the Ross procedure.

BACKGROUND AND AIMS OF THE STUDY: The Ross procedure, in which the aortic valve is replaced with the patient's own pulmonary valve (pulmonary autograft), is considered an excellent alternative for younger patients requiring elective aortic valve replacement. Although resting pulmonary autograft hemodynamics are excellent, exercise hemodynamic data are lacking. The study aim was to measure the hemodynamic performance of the pulmonary autograft with exercise Doppler echocardiography (DE). METHODS: Twenty-four Ross procedure patients (20 males, four females; mean age 46 +/- 11 years) were studied at 25 +/- 14 months after aortic valve replacement with a pulmonary autograft. Patients had baseline supine DE to measure the maximum velocity (Vmax), and the peak and mean pressure gradient across the pulmonary autograft. Effective orifice area was calculated from the continuity equation and indexed to body surface area (EOAi). Patients then underwent symptom-limited upright bicycle exercise with supine DE repeated immediately on stopping exercise. For comparison, 10 normal controls (age 41 +/-10 years) and five mechanical aortic valve patients (mean age 55 +/- 10 years) were studied. RESULTS: At rest: Ross procedure patients had similar Vmax (1.2 +/- 0.2 m/s), peak gradient (6 +/- 2 mmHg), mean gradient (4 +/- 1 mmHg) and EOAi (1.7 +/- 0.4 cm2/m2) to those of normal controls. Mechanical-valve patients had significantly higher Vmax (2.5 +/- 0.2 m/s, p <0.001), peak gradient (25 +/- 4 mmHg, p <0.001) and mean gradient (14 +/- 3 mmHg, p <0.001) than Ross patients and normal controls. At exercise: Ross procedure patients had similar Vmax (1.8 +/- 0.4 m/s versus 2.1 +/- 0.2, p = NS), peak gradient (14 +/- 6 mmHg versus 17 +/- 4, p = NS) and mean gradient (8 +/- 4 mmHg versus 10 +/- 2, p = NS) to normal controls, with no significant change in EOAi. Mechanical-valve patients had significantly higher Vmax (3.4 +/- 0.3, p <0.001), peak gradient (48 +/- 7 mmHg, p <0.001) and mean gradient (30 +/- 5 mmHg, p <0.001) than Ross patients and normal controls. CONCLUSIONS: Aortic valve replacement using the Ross procedure provides excellent hemodynamic results at rest and on exercise, with DE parameters indistinguishable from those of normal controls. This study provides further support for the use of the Ross procedure as a preferred method of aortic valve replacement in younger patients.

Physical mapping of the CXC chemokine locus on human chromosome 4.

A physical map of the CXC chemokine locus on chromosome 4 has been constructed by PCR analysis and PFGE mapping of YAC clones. The genes for IL8, GRO1, PPBP, PF4, SCYB5 (ENA-78) and SCYB6 (GCP-2) have been co-localized on a 335-kb genomic fragment. The GRO2 and GRO3 genes did not map within this region and based on analysis of a YAC contig overlapping IL8 we speculate that GRO2 and GRO3 map downstream of this region. We have also assigned the novel CXC chemokine gene, SCYB9B (alias H174/betaR1) to chromosome 4q21, upstream and within 12 kb of INP10. Like INP10 and MIG, INP10 and SCYB9B are arranged in a head to tail manner. The chromosomal arrangement of these genes appears to reflect the evolution of this multigene family and supports the theory that it arose by gene duplication.

VP4 and VP7 genotyping of rotavirus samples recovered from infected children in Ireland over a 3-year period.

Between September 1995 and August 1998, the incidence and diversity of the main human rotavirus genotypes (G1, G2, G3, and G4 and P[8], P[4], P[6], and P[9]) among Irish children were determined by using established and adapted reverse transcriptase PCR-based genotyping methods. From a total of 193 rotavirus-positive specimens collected from nine hospitals we successfully identified the P type in 182 (94%) of the samples and the G type in 165 (85.5%) of the samples. Only four samples could not be assigned a G or P type. Two P types existed in Ireland, P[8] (78%) and P[4] (16%), and their relative incidence varied over the 3 years of this study. No P[6] or P[9] types were detected. G1 was the most predominant G type (55%), and the incidences of G2, G3, and G4 isolates were 15.5, 1, and 11%, respectively. Three percent of the samples tested had a mixed G type. A P and G type was assigned to 158 (81.8%) of samples. Of the typeable samples, G1 P[8] was the most prevalent (65%), whereas G2 P[4] (17%), G3 P[8] (1%), G4 P[8] (12%), and mixed types (all G1/ G4 P[8]) (4%) were detected less frequently. In the third year a significant genotypic shift from G1 P[8] to G2 P[4] and G4 P[8] was observed. During the study, we noticed that the inclusion of random primers during cDNA synthesis greatly increased the specificity of the PCR typing assays. No correlation was seen between the contributing hospitals and a specific genotype. In conclusion, the coverage of infection given by the recently licensed tetravalent vaccine would be significantly high in Ireland, although future monitoring of genotypic changes among Irish isolates should be encouraged.

Early undifferentiated connective tissue disease (CTD). VI. An inception cohort after 10 years: disease remissions and changes in diagnoses in well established and undifferentiated CTD.

OBJECTIVE: (1) To review the diagnoses after 10 years in patients who were identified within 12 months of the onset of well established and undifferentiated connective tissue diseases (CTD). (2) To examine the death rates and disease remissions in these patients. METHODS: This inception cohort of 410 patients had less than one year of signs and/or symptoms of CTD. Diagnoses of rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and poly/dermatomyositis (PM/DM) were made in 197 patients using accepted diagnostic and classification criteria. Diagnoses of undifferentiated CTD were made in 213 patients. These latter patients were placed in 3 categories: isolated Raynaud's phenomenon (RP), unexplained polyarthritis (UPA), and undifferentiated CTD (UCTD), defined as meeting at least 3 of 11 specific manifestations of CTD. The diagnoses and remissions in all patients after 10 years were determined. RESULTS: Patients with well established CTD tended to remain with the original diagnosis. The 10 year survival was at least 87% in all diagnostic categories, with the exception of SSc, in which it was 56%. The progression of UPA to RA occurred infrequently. The presence of antinuclear antibodies suggested that UPA may develop additional symptoms and/or a specific diagnosis, and RP in these patients increased the likelihood of progressing to UCTD or a specific well established CTD. Ten percent of patients with RP progressed to SSc. In patients with UCTD, joint pain/tenderness and swelling counts were associated with progression to other diagnoses including RA, while either serositis, malar rash, or discoid lupus suggested the eventual diagnosis of SLE. CONCLUSION: The survival of patients with SSc was poor, with most dying early in the course of their disease. Remissions were seen in all groups of patients except SSc. The remissions were sometimes transient in SLE. Undifferentiated disease at initial examination within 12 months of onset usually remains undifferentiated.

Lymphoblast morphology in predicting leukemic meningeal relapse with low chamber count and lymphoblasts.

The diagnostic criteria for meningeal relapse (MR) of acute lymphoblastic leukemia (ALL) are a cerebrospinal fluid (CSF) chamber count of more than five leukocytes per microliter and a cytomorphological evaluation revealing lymphoblasts. A dilemma arises when confronted with a patient with a low CSF white blood cell (WBC) chamber count and lymphoblasts. We utilized a scoring system to review lymphoblast morphology in 12 such patients. A cell was defined as a lymphoblast if it could not be easily categorized as a lymphocyte, monocyte. histiocyte, or granulocyte. Each lymphoblast was scored on four parameters: presence of nucleoli, homogeneous distribution of chromatin, nucleocytoplasmic ratio greater than 75%, and nuclear irregularity. Cells were scored without knowledge of the patients' out come. Seven patients eventually developed MR by current criteria and five patients never relapsed. The mean lymphoblast scores for patients that did and did not relapse were 2.35 and 1.53, respectively (P < .001). The percent of cells scored as lymphoblasts was also significantly higher in patients that relapsed, 36.9% vs. 19.4% (P = .01). Our study shows that careful cytomorphologic analysis can predict which patients with low chamber counts and "blasts" on cytocentrifuge examination will progress to meningeal relapse. We recommend reviewing the definition of MR and using a scoring system when confronted with blasts in a low chamber count cerebrospinal fluid specimen.

Electron arc irradiation of the postmastectomy chest wall: clinical results.

BACKGROUND AND PURPOSE: Since 1980 electron arc irradiation of the postmastectomy chest wall has been the preferred technique for patients with advanced breast cancer at our institution. Here we report the results of this technique in 140 consecutive patients treated from 1980 to 1993. MATERIALS AND METHODS: Thoracic computerized tomography was used to determine internal mammary lymph node depth and chest wall thickness, and for computerized dosimetry calculations. Total doses of 45-50 Gy in 5 to 5 1/2 weeks were delivered to the chest wall and internal mammary lymph nodes via electron arc and, in most cases, supraclavicular and axillary nodes were treated with a matching photon field. Patients were assessed for acute and late radiation changes, local and distant control of disease, and survival. Patients had a minimum follow-up of 1 year after completion of radiation treatment, and a mean follow up interval of 49 months and a median of 33 months. All patients had advanced disease: T stages 1, 2, 3, and 4 represented 21%, 39%, 21% and 19% of the study population, with a mean number of positive axillary lymph nodes of 6.5 (range, 0-29). Analysis was performed according to adjuvant status (no residual disease, n = 90), residual disease (positive margin, n = 15, and primary radiation, n = 2), or recurrent disease (n = 33). RESULTS: Acute radiation reactions were generally mild and self limiting. A total of 26% of patients developed moist desquamation, and 32% had brisk erythema. Actuarial 5 year local-regional control, freedom from distant failure, and cause-specific survival was 91%, 64%, and 75% in the adjuvant group; 84%, 50%, and 53% in the residual disease group; and 63%, 34%, and 32% in the recurrent disease group, respectively. In univariate Cox regressions, the number of positive lymph nodes was predictive for local failure in the adjuvant group (P = 0.037). Chronic complications were minimal with 11% of patients having arm edema, 17% hyperpigmentation, and 13% telangectasia formation. CONCLUSION: These data demonstrate that local-regional control with electron are therapy of the postmastectomy chest wall is comparable to photon techniques. Acute radiation reactions are well tolerated and mostly of minor extent. A previous report demonstrated a significant reduction in the dose-volume relationship of the lung using the electron arc compared with two photon techniques. Consequently, with careful attention to treatment planning and dosimetry, electron arc therapy of the postmastectomy chest wall is safe and effective. The radiation dose to heart and lung is minimized without compromise on local control.

Early undifferentiated connective tissue disease. IV.Musculoskeletal manifestations in a large cohort of patients with undifferentiated connective tissue diseases compared with cohorts of patients with well-established connective tissue diseases: followup analyses in patients with unexplained polyarthritis and patients with rheumatoid arthritis at baseline.

OBJECTIVES: To examine the musculoskeletal manifestations in a large cohort of patients (n = 410) diagnosed with either a well-established connective tissue disease (CTD) (n = 197) or an early undifferentiated CTD (n = 213) with a symptom duration of <1 year. This study was aimed at determining the predictive value of demographic, clinical, and laboratory features on outcome in patients with unexplained polyarthritis (UPA) (from the early undifferentiated CTD cohort; n = 67) or rheumatoid arthritis (RA) (from the well-established CTD cohort; n = 57), over a 5-year followup period. METHODS: Patients from both cohorts were assessed at years 1, 3, and 5. At the study visits, clinical data were collected in a standardized manner, and sera were obtained and stored. A priori criteria were established for patient ascertainment and diagnosis over the duration of the study. Standard statistics were used for comparisons of baseline characteristics in patients diagnosed as having systemic lupus erythematosus, RA, undifferentiated CTD, and UPA at entry into the cohorts. Baseline features in patients with UPA were examined according to the different subsequent outcomes (RA, CTD, or undifferentiated CTD, remission [nonpersistent], or persistent or active UPA). Baseline features in patients with RA whose disease remained active versus those in whom remission was attained were also examined. Two multivariable analyses, classification trees and polychotomous logistic regression, were performed to predict disease outcomes over time. RESULTS: The overall rate of ascertainment for the 410 patients ranged from 90 % at year 1 to 71 % at year 5. Patients with established CTDs showed a tendency for more stable diagnoses than those with early undifferentiated CTDs (90-100% versus 45-70%). Consistent baseline predictors of persistent active disease among patients with RA, in both univariate and multivariable analyses, were higher joint counts for pain and tenderness and higher erythrocyte sedimentation rate (ESR). In approximately 20% of patients who were classified as having RA when they originally entered the cohort, the disease was in remission at 5 years. Twenty percent of the patients originally classified as having UPA developed RA over the duration of the study. These patients tended to be older and to have swelling of small joints at baseline. However, a consistent pattern of predictive variables could not be identified in the multivariable analyses, other than at year 1 (higher small joint counts for swelling and higher ESR). CONCLUSION: Baseline features (joint counts, and ESR) among RA patients were variously predictive of persistently active disease at years 1-5. Consistent baseline predictors of outcome among patients with UPA only emerged at year 1. Remission occurred in approximately 20% of RA patients, whereas a similar percentage of patients with UPA developed RA. These findings have implications with regard to treatment decisions in patients with early RA and/or UPA.

Characterization of a rabbit cationic protein (CAP18) with lipopolysaccharide-inhibitory activity.

Cationic antibacterial proteins (CAP) were purified from rabbit granulocytes, and the effects of CAP on lipopolysaccharide (LPS)-induced tissue factor generation by murine peritoneal macrophages and human blood monocytes were studied. CAP were purified from rabbit peritoneal leukocytes by using as an assay the agglutination of erythrocytes coated with Re-LPS. Two proteins with CAP activity, CAP18 (18 kDa) and CAP7 (7 kDa), were isolated by acid extraction, ethanol precipitation, affinity chromatography, gel filtration, and reverse-phase high-pressure liquid chromatography. On the basis of protein sequencing, CAP7 was identified as the C-terminal fragment of CAP18, designated CAP18(106-142). Various forms of LPS (S-LPS, Re-LPS, and lipid A) activate murine macrophages and human blood monocytes to generate tissue factor (tissue thromboplastin). Incubation of LPS for 18 h with partially purified CAP (heparin-Sepharose fraction) inhibited the capacity of LPS to induce tissue factor; however, purified CAP18 inhibited about 75% of the activity of S-LPS after 1 h of incubation. CAP more effectively inhibited S-LPS than Re-LPS or lipid A. Synthetic CAP18(106-142) inhibited LPS-induced tissue factor generation by murine macrophages. CAP18(106-142) has greater LPS-binding and LPS-neutralizing activities than CAP18. We hypothesize that CAP18 and the derivative peptide, CAP18(106-142), bind to LPS and alter the capacity of LPS to initiate disseminated intravascular coagulation. In this regard, CAP may have therapeutic potential for sepsis and endotoxin shock.

The selective isolation of novel cDNAs encoded by the regions surrounding the human interleukin 4 and 5 genes.

We have developed modifications to direct cDNA selection that allow the rapid and reproducible isolation of low abundance cDNAs encoded by large genomic clones. Biotinylated, cloned genomic DNAs are hybridized in solution with amplifiable cDNAs. The genomic clones and attached cDNAs are captured on streptavidin coated magnetic beads, the cDNAs are eluted and amplified. We have applied this protocol to a 425kb YAC that contains the human IL4 and IL5 genes. After two cycles of enrichment twenty-four cDNAs were evaluated, all of which were homologous to the YAC. DNA sequencing revealed that nine cDNAs were 100% homologous to the interferon regulatory factor 1 (IRF1) gene. Six clones were 70% homologous to the murine P600 gene, which is coexpressed with IL4 and IL5 in mouse Th2 cells. The nine remaining clones were unique within the sequence databases and were non redundant. All of the selected cDNAs were initially present at very low abundance and were enriched by as much as 100,000-fold in two cycles of enrichment. This modified selection technique should be readily applicable to the isolation of many candidate disease loci as well as the derivation of detailed transcription maps across large genomic regions.

Cloning of the cDNA for the serine protease homolog CAP37/azurocidin, a microbicidal and chemotactic protein from human granulocytes.

Human cationic antimicrobial protein (CAP37) is a neutrophil granule protein with monocyte chemotactic and antibacterial activity. A CAP37 cDNA clone of 899 bp was isolated from an HL-60 cDNA library using degenerate oligonucleotide probes based on partial N-terminal sequence of the CAP37 protein. The cDNA sequence predicts an open reading frame of 753 bp encoding a protein of 251 amino acids. A 26-residue eukaryotic signal peptide and a potential 7 amino acid pro-peptide are present at the N-terminus of the protein. The cDNA sequence also predicts three N-linked glycosylation attachment sites and eight intramolecular cysteines. The deduced amino acid sequence of CAP37 shows 44, 42, and 32% homology at the amino acid level to neutrophil elastase, myeloblastin, and cathepsin G, respectively, suggesting that CAP37 is a member of the serine protease gene family. CAP37 does not possess serine protease activity probably due to mutations in two of three residues in the catalytic triad of the "charge relay system." Whereas CAP37 is expressed in undifferentiated HL-60 cells no message is detected in mature neutrophils.

Complementary DNA sequence of rabbit CAP18--a unique lipopolysaccharide binding protein.

CAP18 is a novel 18 kDa cationic protein [pI approximately 10] originally purified from rabbit granulocytes using as an assay the agglutination of lipopolysaccharide (LPS) coated erythrocytes. cDNA clones encoding CAP18 were isolated from a rabbit bone marrow cDNA library using a PCR generated oligonucleotide probe derived from the N-terminal amino acid sequence. The deduced amino acid sequence reveals a putative signal sequence of 29 amino acids and a mature protein of 142 amino acid residues. The predicted size of the encoded protein is 16.6 kDa with a pI of 10. There are no N-linked glycosylation sites. The CAP18 sequence bears no homology with other known LPS-binding proteins including human bacterial permeability increasing protein (BPI)(1) and rabbit LPS binding protein (LBP)(2).

Human neutrophil granule cationic protein CAP37 is a specific macrophage chemotaxin that shares homology with inflammatory proteinases.

Cationic antimicrobial protein CAP37 (Mr = 37 kD) is derived from the azurophilic granules of human PMN. In vitro and in vivo studies demonstrate that CAP37 is a novel monocyte-specific chemoattractant. The N-terminal amino acid sequence of CAP37 shares significant homology with a number of inflammatory molecules with protease activity including elastase and cathepsin G. However, substitutions in the catalytic triad (serine for a histidine at position 41 and glycine for a serine at position 175), may account for its lack of serine protease activity. A full length cDNA for CAP37 was identified in an HL60 cDNA library screened with oligonucleotide probes designed from the N-terminal amino acid sequence. Sequencing of the cDNA reveals a protein of 225 amino acids with significant nucleotide homology to cathepsin G and human neutrophil elastase.

Sp1, a CAAT-binding factor, and the adenovirus major late promoter transcription factor interact with functional regions of the gamma-fibrinogen promoter.

To study the factors which influence the coordinately and developmentally regulated expression of the three adjacent fibrinogen genes, we have defined the functional regions of the gamma-fibrinogen promoter and the proteins which bind to them. Using a series of 5' and internal deletion mutations, we found that sequences between 88 and 43 base pairs (bp) upstream of the gamma-fibrinogen transcription initiation site functioned in cis to direct properly initiated mRNA accumulation in transfected hepatocytes. The efficient function of these sequences was highly distance dependent, since transcriptional activity decreased by 92% when they were moved 32 bp upstream of the TATA box. We demonstrated that two known and one putative transcriptional factors interacted with this 47-bp sequence. The transcription factor Sp1 interacted with sequences between -51 and -46 as demonstrated by protection from DNase I digestion with the purified protein. Directly adjacent to the Sp1 site, between nucleotides -66 and -53, there was a sequence which bound a CAAT-binding factor. Finally, sequences just 5' to the CAAT factor-binding site interacted with the adenovirus major late transcriptional factor as previously demonstrated. Internal deletion mutations which disrupt these interactions diminished the activity of the promoter in vivo. One consequence of the interaction of these proteins is that a bend is placed in the DNA at or near their sites of interaction.