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We measured the concentrations of total porphyrins and their metabolites (uro-, hepta-, hexa-, penta-, copro- and protoporphyrin) in various human tissues: liver, erythrocytes, skin, adipose tissue, and mammary gland. The porphyrin concentrations varied within major limits, e.g., 3.1 +/- 2.3 nmol porphyrins/g liver and 0.50 +/- 0.10 nmol/g erythrocytes. No significant differences were detectable in other tissues in comparison with liver. In all tissues, the predominant metabolite was protoporphyrin, followed by coproporphyrin, whereas only low concentrations of higher carboxylated porphyrins such as uroporphyrin were detectable. It is concluded that porphyrin metabolism and its regulation is similar in all human tissues, perhaps with some small differences in the erythrocytes.

The two 5'-proximal open reading frames of peanut clump virus RNA 2, which encode the coat protein of 23 kDa and a protein of 39 kDa (P39), are both translated in vitro from genomic RNA 2. We have studied the translational strategy involved in the initiation at the second AUG of RNA 2, which is the initiation codon of P39. Mutation experiments with synthetic transcripts corresponding to the 5'-half of RNA 2 ruled out mechanisms of P39 translation initiation involving termination-reinitiation and internal ribosomes entry. The results were consistent, however, with a leaky scanning mechanism for P39 initiation, in which about one-third of the ribosomes fail to initiate translation of coat protein and scan along the template to initiate translation at the AUG of the P39 gene, more than 1000 residues in from the 5'-terminus of the RNA 2.

Translation of tomato black ring virus (TBRV) RNA-1 in a rabbit reticulocyte lysate leads to the synthesis of a 250K polyprotein which cleaves itself into smaller proteins of 50, 60, 120, and 190K. Polypeptides synthesized from synthetic transcripts corresponding to different regions of TBRV RNA-1 are processed only when they encode the 23K protein delimited earlier by sequence homology with the cowpea mosaic virus 24K protease. The proteolytic activity of this protein is completely lost by mutating residues C170 (to I) or L188 (to H), residues which align with conserved residues of the viral serine-like proteases. The 120K protein is generated by cleavage of the dipeptide K/A localized in front of the VPg but is not further cleaved in vitro at the K/S site (at the C terminus of the VPg) or between the protease and polymerase domains. However, both the protein VPgProPol (120K) and the protein ProPol (117K) produced in vitro from synthetic transcripts can cleave in trans the RNA-2-encoded 150K polyprotein, but they cannot cleave in trans polypeptides containing a cleavage site expressed from RNA-1 transcripts in which the protease cistron is absent or modified.

The cyclin D1 gene, located on chromosome 11q13, is frequently rearranged in parathyroid neoplasms and amplified in some carcinomas of other organs. Recent studies have detected amplification of cyclin D1 and other markers on chromosome 11q13 (evaluated by Southern or slot blot assays) in approximately 25-50% of squamous cell carcinomas of the esophagus and noted that amplification was associated with lessened survival time. We applied the technique of differential polymerase chain reaction to the evaluation of cyclin D1 gene amplification in squamous cell carcinomas of the esophagus. Cyclin D1 was found to be amplified in 10 of 45 (22%) primary tumors and three of 12 (25%) lymph node metastases. Lymph node metastases tended to be more common in patients with cyclin D1 amplification (70%) than in those without amplification (37%). In 36 patients with follow-up, cyclin D1 amplification was associated with decreased 1 year survival (28% vs. 59%). Cyclin D1 gene amplification in esophageal carcinomas can be evaluated by differential polymerase chain reaction and may provide useful prognostic information.

The complete nucleotide sequence of RNA 1 of the tentative furovirus peanut clump virus (PCV) has been determined by characterization of cloned cDNA and by direct RNA sequencing. The sequence is 5897 nucleotides in length and contains three long open reading frames (ORFs). The 5'-terminal proximal ORF has the potential to encode a polypeptide of M(r) 130942 (P131) containing methyltransferase and RNA helicase homologous domains and displaying homology with large nonstructural proteins of alpha-like viruses, which are known or thought to be involved in virus replication. The P131 ORF is followed in-frame by a second ORF which is probably expressed by partial readthrough of the UGA termination codon of the P131 ORF to produce a polypeptide of M(r) 191044 (P191). The readthrough region of P191 contains the characteristic 'core' RNA polymerase motif, indicating that the PCV replicase proteins are expressed as a pair of overlapping proteins as in the tobamoviruses, tobraviruses and the furovirus soil-borne wheat mosaic virus (SBWMV). Sequence comparisons indicate that P131 and P191 are most closely related to the replicase proteins of SBWMV and the hordeivirus barley stripe mosaic virus (BSMV) but are only distantly related to the replicase of the furovirus beet necrotic yellow vein virus (BNYVV). The 3'-terminal proximal ORF can encode a putative polypeptide of M(r) 14556 (P15) which displays homology to small cysteine-rich proteins of hordeiviruses and SBWMV. We have corrected four errors in the sequence of PCV RNA 2 published previously by Manohar et al. (Virology 195, 33-41, 1993). One of these changes causes two small ORFs near the 3' terminus of RNA 2 to be fused together to create an ORF for a putative polypeptide of M(r) 16833 (P17) which displays extensive homology with the third protein of the triple gene block of BSMV RNA beta.

Previous studies have found that amplification and overexpression of the c-erbB-2 oncogene in mammary ductal adenocarcinomas predicts decreased disease-free or overall survival. Information regarding the prognostic and pathogenetic significance of oncogene amplification has been limited by difficulty obtaining sufficient quantities of high molecular weight DNA for Southern blot analysis. Differential polymerase chain reaction (PCR) has been suggested as an alternative method for evaluating gene amplification and can be performed using formalin-fixed paraffin-embedded specimens. The authors of this study used differential PCR to detect c-erbB-2 gene amplification and immunohistochemistry to evaluate c-erbB-2 expression. A highly significant degree of concordance (P < .002) between c-erbB-2 amplification and expression was observed. Abnormalities of c-erbB-2 copy number or expression were more common in tumors with higher histologic grade, and trends were noted toward association with other prognostically unfavorable biologic markers, such as reduced progesterone receptor content and DNA aneuploidy.

Previous studies have demonstrated quantitation of epidermal growth factor receptors (EGFR) to be of prognostic significance in breast, bladder, esophageal and other neoplasms. However, the relatively large quantity of unfixed tissue required for epidermal growth factor radioligand binding assays (RLBA) has precluded its application to cytologic specimens and small biopsy specimens. For this reason we evaluated reverse transcription intron differential polymerase chain reaction (RTIDPCR) as an assay of EGFR gene expression. Squamous cell carcinoma (A431 and SiHa), transitional cell carcinoma (HT1376, T24, RT4), mammary (MCF7) and endocervical (HeLa) adenocarcinoma, and leukemia (K562) cell lines were used to compare RTIDPCR and RLBA. RTIDPCR involved reverse transcription of RNA and amplification of cDNA using primers for beta-actin and EGFR. Good agreement was observed between the RLBA and RTIDPCR results. RNA extracted from fresh cells, Diff-Quik-stained smears and formalin-fixed, paraffin-embedded cell pellet sections yielded similar results. These data suggest that RTIDPCR may be useful in evaluating gene expression by cells processed as cytologic specimens.

The composition and antacid properties of six samples of codried antacids containing both aluminum and magnesium were determined. Aluminum hydroxide-magnesium carbonate codried gel and aluminum hydroxide-magnesium hydroxide codried gel were non-homogeneous, as the samples contained combinations of hydrotalcite, amorphous aluminum hydroxide, magnesium hydroxide, magnesium hydroxycarbonate, and magnesium carbonate. All samples passed the preliminary antacid test and had high acid neutralizing capacities. However, the rate of acid neutralization varied between samples. In some cases the rate of acid neutralization at a dose of 400 mg was too slow to raise the pH to 3.0 as required by the Rossett-Rice test.

The composition and antacid properties of 10 samples of crystalline antacids containing both aluminum and magnesium were determined. The composition was found to vary significantly, even within the same type of antacid. For example, three of four hydrotalcite samples exhibit evidence of the presence of a minor phase of amorphous aluminum hydroxide. Almagate and almagcit, which are claimed to be unique compounds, were found to be composed of hydrotalcite, magnesium hydroxycarbonate, and/or magnesium carbonate and amorphous aluminum hydroxide. All three magaldrate samples examined contained a minor phase of amorphous aluminum hydroxide. All 10 samples passed the preliminary antacid test and had high acid neutralizing capacities. However, the rate of acid neutralization varied between samples. In some cases the rate of acid neutralization at a dose of 400 mg was too slow to raise the pH to 3.0 as required by the Rossett-Rice test.

Tomato black ring virus isolate L supports the multiplication of a large satellite RNA of 1376 nt which has no common features with the two genomic RNAs except for the terminal motif 5' VPg UUGAAAA and a 3' poly(A) tail. The TBRV sat-RNA contains an ORF for a protein of 48K which is translated both in vitro and in vivo. To determine the function of the 48K protein we have studied the effect of different mutations introduced in the ORF of the cDNA clone on the capacity of transcripts to multiply in Chenopodium quinoa plants or protoplasts when inoculated along with the genomic RNAs. Transcripts in which nucleotides have been substituted within the 5' proximal region of the ORF multiplied poorly even when the modification conserved the 48K protein sequence, suggesting that this portion of the ORF contains cis-acting RNA sequences. Transcripts with alterations in the internal region of the ORF retained their multiplication capacity provided the mutation did not destroy the ORF or modify the length of the protein expressed. The absence of multiplication in plants of transcripts unable to express the 48K protein and their inability to replicate in protoplasts suggest strongly that the sat-RNA translation product itself is implicated in the replication of sat-RNA.

Satellite RNA depend for their multiplication on the co-infection of a host cell by a helper virus which can itself multiply independently of the satellite. Four types of satellite RNA have been distinguished on the basis of the size of the RNA and what sort, if any, of protein they encode. One of them, the B-type, comprises relatively large RNA which are messenger RNA for non-structural proteins. Many of these satellites are typified by having nepoviruses as helper viruses. In general, the presence of nepovirus mRNA satellites in a virus culture causes little or no modification to the symptoms of infection by the helper virus and has little effect on its yield. Some satellites appear to be highly specific to a strain of helper virus but others can be helped by heterologous viruses. The proteins encoded by nepovirus mRNA satellites have a M(r) of 38,000 to 48,000 and are relatively basic, in particular in the N-terminal and C-terminal parts of the molecules. However, there is little similarity in amino acid sequence between proteins encoded by different satellites and no peptide motif could be found in all satellite proteins. The results of reverse genetics experiments with satellites suggest that the satellite-encoded protein is essential for the multiplication of the satellite RNA. This system has considerable potential for the study of the mechanisms of replication both of satellite and helper virus RNA.

The synthesis of proteins encoded by the RNA of tomato black ring virus (TBRV) in vivo was studied in protoplasts by direct labelling with [35S]methionine, and in protoplasts and plants by immunoblotting experiments with specific antisera. Comparison of the proteins synthesized in infected and mock-inoculated protoplasts suggested that proteins of M(r) 120K, 90K, 80K, 57K and 46K were virus-specific. The proteins derived from the RNA-1-encoded polyprotein detected by immunoblotting were a stable 120K protein and, only in protoplasts, small amounts of a 90K protein which contains the C-terminal part of the 120K protein and the polymerase domain. The results suggest that the polymerase and the adjacent protease function in vivo largely or solely when combined in a 120K protein. The proteins derived from the RNA-2-encoded polyprotein detected by immunoblotting were 59K and 57K proteins, which reacted with antiserum to TBRV particles, and a 46K protein. In extracts of infected Nicotiana clevelandii and Chenopodium quinoa made soon after inoculation, the 59K protein was more abundant than the 57K protein; later samples contained similar quantities of each protein. The 57K protein comigrated with protein extracted from virus particles. The results of amino acid sequencing suggested that the 57K protein is derived from the 59K protein by the loss of nine C-terminal amino acids. Antiserum to a peptide adjacent to the 57K protein in the 150K polyprotein detected a 46K protein in protoplasts and plant tissue. The results support the processing scheme for TBRV polyproteins proposed after analysis of the products of in vitro translation.

We determined the effects of epidermal growth factor, insulin-like-growth-factor-1 and estradiol on the anchorage independent growth of the estrogen receptor positive human breast cancer cell lines MCF7 and T-47D. In serum free conditions growth factors but not estrogen induced a dose dependent stimulation of growth in both cell lines. The ability of estrogen to induce colony formation of early passage MCF7 cells (less than 100) was strictly correlated to the concentration of sulfatase and charcoal treated calf serum (CCS) with a maximal effect at a concentration of 5% CCS and 10 nM estradiol. CCS alone had no stimulatory effect on the anchorage independent growth of early passage MCF7 cells, but increased colony formation in late passage (greater than 1000) MCF7 and T-47D cells. The growth of late passage MCF7 cells was inhibited by antiestrogen. Thus, the presence of serum components is necessary for the effect of estrogen but not for the effects of growth factors on the anchorage independent growth of estrogen receptor positive human breast cancer cell lines; after a prolonged period of tissue culture serum components switch their function from indirectly modulating estrogen effects to directly stimulating growth in the absence of estrogen.

Management of lymphoedema is extremely variable in the medical profession, ranging from total denial of diagnostic procedures and treatment to excessively aggressive treatments. Between these 2 extremes lies the manual lymphatic drainage technique according to Földi, which is an non-invasive physiological method. In some cases, it may be necessary to associate the use of sequential pneumatic compression or the introduction of medicaments of the benzopyrone family. Due to this approach, the functional limitations and long term complications are well controlled. The results depend, of course, on the patients willingness to follow the compressive treatment for the rest of his life.

In vitro translation of RNA-2 of each of two closely related nepoviruses, tomato black ring virus (TBRV) and grapevine chrome mosaic virus (GCMV), in a rabbit reticulocyte lysate resulted in the synthesis of single polypeptides of 150K and 146K respectively. Processing of these polyproteins occurred after the addition of translation products of homologous RNA-1. The positions of the cleavage products within the polyproteins were determined. From the N to the C terminus, Mr values for the proteins were 50K, 46K and 59K for TBRV and 44K, 46K and 56K for GCMV. TBRV RNA-1 translation products also cleaved the polyproteins encoded by GCMV RNA-2 which suggests that the cleavage sites in the two polyproteins are similar.

In an effort to explore the use of polypeptide growth factors as potential markers for cancer detection, we have identified the presence of transforming growth factor-alpha (TGF-alpha) in pooled urine of patients with metastatic breast cancer by a commercial radioimmunoassay (RIA) based on a rabbit antiserum raised to the C-terminal 17aa synthetic fragment of rat TGF-alpha. This TGF-alpha RIA detected both high molecular weight (HMW) and low molecular weight (LMW) forms of TGF-alpha in the conditioned media of a breast cancer cell line (MDA-MB-231) and in the urine of healthy women and those with breast cancer. The ratio of HMW to LMW species of TGF-alpha by RIA after Bio-Gel P-100 chromatography was approximately equal in pooled urine samples from both healthy women and those with breast cancer, and in the conditioned media from the cell line MDA-MB-231. Using established procedures for concentrating urinary proteins from 24-h urine samples by adsorption onto methyl-bonded microparticulate silica and selective elution by acetonitrile, TGF-alpha RIA results from women with disseminated breast carcinoma were compared with those of healthy pre- and post-menopausal control women. Analysis indicated a median TGF-alpha value of 981 ng/g urinary creatinine for urine samples from cancer patients (range 608 to 1737) and 642 ng/g creatinine (range 417 to 941) for control urine samples. Although the difference was statistically significant (p less than 0.05), urinary TGF-alpha detection with this assay method appears to have limited usefulness as a diagnostic marker for metastatic human adenocarcinoma of the breast.(ABSTRACT TRUNCATED AT 250 WORDS)

Tomato black ring virus RNA-1 was translated in a rabbit reticulocyte lysate. The primary translation product of Mr 250K, which corresponds to its whole coding capacity, was synthesized within 45 min and, during further incubation in the translation medium, was proteolytically processed. Essentially, four cleavage products (P190, P120, P60 and P50) were detected and located within P250 by pulse-chase and immunoprecipitation experiments. P190 is an intermediate cleavage product which is further cleaved to form P60 and P120. P120, which contains the region that has been assigned to the virus protease and the virus polymerase, was not further cleaved in vitro.

Synthetic transcripts of tomato black ring virus satellite RNA (TBRV satRNA), isolate L, were prepared from cDNA cloned in the Bluescribe transcription vector. Transcripts with 49 (T49L) or two (T2GL) extra nucleotides at their 5' ends and 42 extra nucleotides at their 3' ends were able to induce, but to different extents, the synthesis in vitro of the satRNA-encoded 48K protein. However, when inoculated into Chenopodium quinoa together with TBRV L genomic RNAs, only T2GL was biologically active, in the presence or absence of a 5' cap analogue in the transcription reactions. Analysis of the 5' and 3' termini of the satRNA isolated from plants showed that nonviral extensions were not maintained in the transcript progeny.

A hybridization probe was used to study the regulation of expression of the gene coding for the small subunit of ribulose 1,5-bisphosphate carboxylase, during functional differentiation of protoplasts. A library of cDNA from poly(A)-containing RNA extracted from specially treated tobacco leaves was constructed in the plasmid pBR322 by blunt-end ligation. This library was screened by colony hybridization with 32P-labelled cDNA prepared from mRNA coding for the precursor of the small subunit. A positive colony was identified containing recombinant plasmids with a nucleotide sequence homologous to this mRNA. These plasmids, bound to diazobenzyloxymethylated cellulose paper, were then used as a hybridization probe. The results showed unambiguously that the small subunit was not transcribed in protoplasts but was transcribed in undifferentiated white and chlorophyll-containing green callus cultures derived from protoplasts. The discrepancy between these results and those obtained with classical techniques is discussed.

On centrifugation in a CsCl density gradient the three tymoviruses, eggplant mosaic, wild cucumber mosaic, and okra mosaic, separate into a major bottom component and several less dense minor components. The RNA of the top component is composed of about 90% tRNA and 10% of an approximately 250 000 dalton messenger RNA. The latter induced the synthesis of coat protein when translated in wheat-germ and rabbit-reticulocyte cell-free systems.

The influence of a heavy soluble dicaprylate salt preparation of a new trypanocidal diamidine (DiaPBF) on systolic and diastolic blood pressure and heart rate of rats has been compared with the effects of the easily soluble DiaPBF-dihydrochloride (DiaPBF-diHCl). After the dicaprylate the drop of the systolic blood pressure was 50% smaller than after an equimolar doses of DiaPBF-diHCl.

Studies of proteins synthesized in vitro by messenger RNA (mRNA) extracted from tobacco protoplasts showed that the changes in protein synthesis and especially the lack of certain proteins observed previously in isolated protoplasts did not result from a failure of translation.

RNA 3 of alfalfa mosaic virus (AlMV) contains information for two genes: near the 5' end an active gene coding for a 35 Kd protein and, near the 3' end, a silent gene coding for viral coat protein. We have determined a sequence of 318 nucleotides which contains the potential initiation codon for the 35 Kd protein at 258 nucleotides from the 5' end. This long leader sequence can form initiation complexes containing three 80 S ribosomes. A shorter species of RNA, corresponding to a molecule of RNA 3 lacking the cap and the first 154 nucleotides (RNA 3') has been isolated. The remaining leader sequence of 104 nucleotides in RNA 3' forms a single 80 S initiation complex with wheat germ ribosomes. The location of the regions of the leader sequence of RNA 3 involved in initiation complex formation with 80 S ribosomes is reported.

RNA 4 of alfalfa mosaic virus (AMV) is a monocistronic messenger for the coat protein. We have determined the sequence of the 40 +/- 2 nucleotides in RNA 4 that were protected in the initiation complex formed with wheat germ 80 S ribosomes from digestion by T1 or pancreatic ribonucleases. The AUG coat protein initiation codon was near the middle of this protected region. We have found two ribosome-binding sites in RNA 3. The principal one, near the 5' end, is the initiation site for the major translation product, a 35,000 dalton protein. The second site binds ribosomes only weakly, at the beginning of the "silent" coat protein cistron, and is similar but not identical to the initiation site protection site is discussed.

In vitro translation of tomato bushy stunt (TBSV)-RNA in a rabbit reticulocyte system resulted in synthesis of five proteins P 18, P 25, P 34, P 35, and P 40. The P 40 protein was identified as the viral coat protein. Fractionation of TBSV-RNA and subsequent translation provided evidence for the existence of discrete subgenomic RNAs.

The products synthesized in vitro by messenger RNA (mRNA) extracted from Nicotiana sylvestris were analyzed by electrophoresis on polyacrylamide slab gels. Only three of the major polypeptides synthesized are considered here: P55, P32, and P20. P55 and P32 were translated from chloroplast mRNA. P55 corresponds to the large subunit of ribulose-1,5-bisphosphate (RuP2) carboxylase; P32 is probably a chloroplast membrane protein. P20, the polypeptide synthesized from cytoplasmic poly(A)(+) RNA, is the precursor of the small subunit of RuP2 carboxylase. The balance between P20 and P32, in which their relative proportions varied inversely, was regulated by the age of the leaves and the time of illumination; we took advantage of this phenomenon to isolate the mRNA from the small subunit in relatively large amounts. This mRNA has a molecular weight of 350,000.

Turnip yellow mosaic virus RNA can be separated into two distinct components of 2 times 10(6) and 300 000 daltons molecular weight after moderate heat treatment in the presence of SDS or EDTA. The two species cannot have arisen by accidental in vitro degradation of a larger RNA, as they both possess capped 5' ends. Analysis of the newly synthesized proteins resulting from translation of each RNA by a wheat germ extract shows that the 300 000 molecular weight RNA can be translated very efficiently into coat protein. When translated in vitro the longer RNA gave a series of high molecular weight polypeptides but only very small amounts of a polypeptide having about the same mass as the coat protein. Thus our results suggest that the small RNA is the functional messenger for coat protein synthesis in infected cells.