[Abnormalities in tattooed skin: sometimes sarcoidosis]. / Afwijkingen in getatoeëerde huid: soms sarcoïdose.

A 43-year-old man presented with a nodular tattoo lesion on his right upperarm. Histologically it resembled the granulomatous reaction seen in systemic sarcoidosis. Further evaluation revealed asymmetrical hilar lymphadenopathy with no interstitial lung disease. Since the patient was a heavy smoker, bronchus carcinoma could not be excluded and cervical mediastinoscopy was performed in order to obtain a lymph-node biopsy. This confirmed the diagnosis of systemic sarcoidosis. The patient was treated by local application of corticosteroids, but with little result. Skin lesions in scars or tattoos may be the first symptom of systemic sarcoidosis. Skin biopsy for histological confirmation of the diagnosis is recommended, as is further investigation to evaluate other organ systems which may be affected.

Development and characterization of a bispecific single-chain antibody directed against T cells and ovarian carcinoma.

Bispecific antibodies with specificity for tumor antigen and CD3 have been shown to redirect the cytotoxicity of T cells against relevant tumor. Our objective was to generate single-chain bispecific antibodies (bsSCA) that could retarget mouse cytotoxic T lymphocytes (CTL) to destroy human ovarian carcinoma in a xenogeneic setting. A bsSCA, 2C11 x B43.13, was constructed by genetic engineering and expressed in mammalian cells. Molecular characteristics, binding properties, and ability to retarget CTL were studied. Western blot analysis showed that the product is a 65-kDa protein. Purification of antibodies could be done by single-step affinity chromatography using protein L-agarose with an unoptimized yield of 200 microg/L. BsSCA 2C11 x B43.13 was capable of binding to mouse CD3 and human CA125 as detected by FACS analysis of EL4 and OVCAR Nu3H2 cells, respectively. It could also bridge activated splenic T cells and human ovarian carcinoma as demonstrated by a bridge FACS assay. Redirected mouse CTL could mediate human target cell lysis in a 20-h 51Cr release assay despite that they are xenogeneic. Prolonged incubation of redirected CTL and tumor targets resulted in a dramatic reduction in tumor cell number. CD28 co-stimulation enhanced redirected CTL function in both types of assays. BsSCA 2C11 x B43.13 thus can be used as a preclinical immunotherapeutic model for human ovarian cancer in a xenogeneic setting.

Pharmacokinetics of aerosolized tobramycin in adult patients with cystic fibrosis.

This study was performed to determine the clinical pharmacokinetics of tobramycin in six patients with cystic fibrosis (CF) after inhalation of 600 mg. Tobramycin was administered with an ultrasonic nebulizer (WISTO SENIOR). Blood and urine were sampled until 24 h after inhalation. Maximum tobramycin levels in serum varied from 0.19 to 2.57 mg/liter (mean 1.27 mg/liter; standard deviation, 1.07 mg/liter). Systemic availability (calculated from urinary output) ranged from 6.0 to 27.4% (mean, 17.5%; standard deviation, 8.8%). The results illustrate that, provided that the systemic availability of tobramycin is a reflection of pulmonary deposition, inhalation studies with CF patients should have a concentration-controlled design. Furthermore, reliance on dose recommendations from the literature for a new patient starting on this treatment is not justified, but it is mandatory that deposition kinetics be studied for each patient and for each nebulizer. It may well be that, with higher levels of deposition, dosages lower than those recommended in the literature will suffice to obtain the desired clinical effect. In addition, the reverse may also be the case.

Rat liver is not damaged by high dose tryptophan treatment.

Adult male Sprague-Dawley rats were treated with USP-grade L-tryptophan at a level of 250 mg/kg seven times over 14 d or three times over 3 d by gastric gavage. At autopsy liver specimens were prepared for histological study by stains specific for lipids, for glycoprotein and glycogen, and for fine structure by electron microscopy. Liver lipid did not accumulate as a result of tryptophan treatment. In a series of unfed animals, however, liver lipid had accumulated within 24 h of food withdrawal. Tryptophan has been implicated in fatty liver development by several reports that cite each other, but, in all cases but one, unfed animals were used, and the data show that liver lipid was already present in the unfed animals at the beginning of the experiment. Tryptophan has also been cited as causing abnormal liver morphology, but our evidence suggests that such observations are the result of artifact induced by frozen section preparation and not the result of tryptophan treatment. Our experiments indicate that tryptophan administered to rats at dosages in excess of those recommended for humans does not induce fatty liver or other morphological changes detectable by the methods described.

Effect of Exogenous Indole-3-Acetic Acid and Indole-3-Butyric Acid on Internal Levels of the Respective Auxins and Their Conjugation with Aspartic Acid during Adventitious Root Formation in Pea Cuttings.

The influence of exogenous indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) on the internal levels of these auxins was studied during the first 4 days of adventitious root formation in cuttings of Pisum sativum L. The quantitations were done by high performance liquid chromatography with spectrofluorometric detection. IBA, identified by combined gas chromatography-mass spectrometry (GC-MS), was found to naturally occur in this plant material. The root inducing ability of exogenous IBA was superior to that of IAA. The IAA level in the tissue increased considerably on the first day after application of IAA, but rapidly decreased again, returning to a level twice the control by day 3. The predominant metabolic route was conjugation with aspartic acid, as reflected by the increase in the level of indole-3-acetylaspartic acid. The IBA treatment resulted in increases in the levels of IBA, IAA, and indole-3-acetylaspartic acid. The IAA content rapidly returned to control levels, whereas the IBA level remained high throughout the experimental period. High amounts of indole-3-butyrylaspartic acid were found in the tissue after feeding with IBA. The identity of the conjugate was confirmed by (1)H-nuclear magnetic resonance and GC-MS. IBA was much more stable in solution than IAA. No IAA was detected after 48 hours, whereas 70% IBA was still recovered after this time. The relatively higher root inducing ability of IBA is ascribed to the fact that its level remained elevated longer than that of IAA, even though IBA was metabolized in the tissue. Adventitious root formation is discussed on the basis of these findings.

Expression of a Neurospora crassa metallothionein and its variants in Escherichia coli.

The Neurospora crassa metallothionein (NC) synthesis gene was cloned and expressed in Escherichia coli in two different expression vectors (pING2 and pUA7), both under the regulation of the Salmonella typhimurium arabinose operon. Upon induction with arabinose, the pING2-NC vector expressed as inclusion body-localized AraB'::NC fusion protein of 21 kilodaltons. The pUA7-NC vector expressed a 5.3-kilodalton Lpp::NC fusion protein anchored to the outer membrane of the cell. Cells expressing the NC fusion proteins accumulated Cd2+ and Cu+ (between 2.3- and 11-fold) compared with nonexpressing cells. To generate novel forms of metal-binding peptides, a set of specific mutant genes for N. crassa NC was designed in which each cysteine residue was replaced with a subset of amino acids implicated in peptide-metal coordination (Asn, Asp, His, Lys, or Tyr residues). These mutant NC sequences were cloned into the two vectors and expressed in E. coli. One of the mutant proteins (containing His residues) showed accumulation of Cd2+ and Cu+ (threefold) from a mixture of 16 heavy metals species. None of the other heavy metals present in the culture was accumulated.

Human metallothionein-II is synthesized as a stable membrane-localized fusion protein in Escherichia coli.

A synthetic gene encoding human metallothionein-II (HMT) was cloned into the specially constructed high-copy-number expression vector, pUA7, and expressed in Escherichia coli. The plasmid construct includes the promoter/operator and regulatory sequences of the Salmonella typhimurium ara operon and part of the 5'-coding and all of the 3'-noncoding regions of the E. coli lpp. Upon induction with arabinose, the resulting Lpp::HMT fusion protein was produced 75,000-fold over uninduced cells, with a relatively stable mRNA (T1/2 of 8.3 min) and a completely stable protein. In addition, over 95% of the final fusion protein was localized in the outer membrane and was capable of binding heavy metals (especially cadmium) in vitro. Cells producing Lpp::HMT bioaccumulated heavy metals (e.g., cadmium) 66-fold over nonproducing cells.

Intraluminal chemistry of zinc in milks.

The solubility of both free and low molecular weight ligand complexed calcium, magnesium, and zinc in skimmed human and bovine milks over intestinal luminal pH ranges (approximately 3-7) was measured using ultrafiltration techniques. Some of the experimental difficulties associated with the separation of labile metal ion ligand components from milks by ultrafiltration techniques are discussed. Experimental methods designed to minimize interferences in mineral ultrafiltrations from milks are outlined. Mineral solubilities in skimmed human and bovine milks are compared to data obtained in a previous study using milk models. The solubility of zinc in both skimmed bovine and bovine model milks is less than in human and human model milks at the higher pHs, characteristic of the luminal region where zinc absorption is thought to occur. The decrease in zinc solubility is caused by the coprecipitation of zinc with calcium phosphate, particularly in bovine milk samples. If solubility at the higher pHs is a requisite for zinc absorption then the enhanced bioavailability of zinc from human milk may be related to the detrimental element-compound interaction discussed in this study.

Coprecipitation modulates the solubility of minerals in bovine milk.

The solubilities of zinc, iron, copper, magnesium, calcium, inorganic phosphate, and citrate in milk decreased when acidic milk preparations were neutralized. In decaseinated bovine milk soluble zinc, iron, and copper were reduced 90%, 60%, and 50%, respectively, as the pH was raised from 4 to 7. Simultaneous precipitation of minerals and citrate was confirmed by analysis of washed precipitate. We propose that the diminished solubilities of zinc, iron, copper, magnesium, and citrate are linked to the precipitation of calcium phosphate through one or more mechanisms of coprecipitation. Such control on mineral solubility may have an impact upon mineral absorption from milk.

Several nodulins of soybean share structural domains but differ in their subcellular locations.

Four soybean cDNA nodule-specific clones encoding nodulin-23, -26b, -27 and -44 were observed to cross-hybridize under low stringency conditions. Nucleotide sequence analysis revealed that the cDNAs contain three distinct domains: two domains with 70 to 95% homology separated by a third domain unique to each cDNA. Despite a number of nucleotide insertions and deletions, the protein sequences are conserved in the two domains which correlate with the homologous nucleotide domains. The amino terminal domain of each nodulin contains putative signal sequences for membrane translocation, although only two (nodulin-23 and -44) meet all the criteria for a functional signal. Immuno-precipitation of hybrid-release translation products of the four cDNAs revealed that nodulin-23 is associated with the peribacteroid membrane while nodulin-27 is in the cytoplasmic fraction of the nodule. These four nodulins are members of a diverse family with conserved structural features and the genes encoding them appear to have recently evolved from a common ancestor.

Effect of pH on the speciation and solubility of divalent metals in human and bovine milks.

Computer models estimated the ligand speciation and solubility of calcium, magnesium, zinc, and copper over a pH range for low molecular weight fractions characteristic of either human or bovine milks. Above pH 4 calcium is the only metal predicted to precipitate. Most of the remaining soluble calcium, magnesium, and zinc should be complexed with citrate. The solubility of calcium, magnesium, and zinc in human and bovine milks was measured experimentally from pH 2 to 7. The solubility of all three metals decreased as the pH increased. Calcium and zinc were soluble over a narrower pH range in bovine milk than in human milk. Increasing the levels of either calcium or inorganic phosphate alone in decaseinated human milk did not affect the solubility of zinc, but when both calcium and inorganic phosphate were added at levels comparable to bovine milk the solubility of zinc decreased at the higher pH's. The decreased solubility of zinc in skimmed milks in pH's characteristic of the small intestine is likely due to coprecipitation of zinc with calcium phosphate--a reaction not predicted for milk systems from known chemical solubility product data.

Stability of histone mRNAs is related to their location in polysomes.

Synthesis of histone mRNAs is closely coupled to DNA synthesis. Following inhibition of DNA synthesis in L6 myoblasts with cytosine arabinoside, a coordinate and exaggerated rate of degradation of histone mRNAs occurs while other mRNAs, encoding ribosomal protein L32 and actin, are unaffected. Inhibition of protein synthesis by puromycin, emetine, or cycloheximide stabilizes histone mRNAs and results in their accumulation. When inhibition of DNA synthesis was followed immediately by inhibition of protein synthesis, the exaggerated rate of decay of the existing subspecies of histone H4 mRNAs was prevented and histone mRNA accumulated. If inhibition of protein synthesis was delayed longer than 3 minutes following inhibition of DNA synthesis, the ability to accumulate H4 mRNAs was lost. Furthermore, new protein synthesis was required to activate the mechanism which specifically destabilized histone mRNA. Puromycin was able to prevent the exaggerated rate of degradation of the various subspecies of H4 mRNA when added up to 15 min after inhibition of DNA synthesis, whereas emetine was effective only when added up to 5 min following inhibition of DNA synthesis. These data suggest that histone H4 mRNAs in polysomes are better targets than those released from polysomes for the specific mechanism which destabilizes histone mRNAs upon inhibition of DNA synthesis.

Low molecular weight copper-binding ligands in human bile.

The aim of this study was to detect and identify major low molecular weight (less than 10,000) copper-binding ligands in human bile. Modified gel chromatography was used as the method of ligand detection because it ensures the detection of labile as well as inert metal-ligand complexes. Conjugated bilirubin, peptides, and amino acids, primarily glycine, were isolated as the major ligands. In contrast to the other copper-binding ligands, the peptides were poor zinc binders, suggesting the possibility that they may confer necessary specificity to trace metal elimination.

Specific mRNP complexes. Characterization of the proteins bound to histone H4 mRNAs isolated from L6 myoblasts.

These studies were designed to identify the proteins associated with specific mRNAs. L6 myoblasts contain a unique poly(A)-rich H4 mRNA as well as poly(A)-minus H4 mRNA subspecies. We have characterized the proteins present in both poly(A)-rich and poly(A)-minus histone H4 mRNP complexes following ultraviolet cross-linking in vivo. In addition, the muscle-specific myosin heavy chain (MHC) mRNP complex was characterized in myoblasts. [35S]Methionine-labelled poly(A)-rich and poly(A)-minus RNP complexes were prepared from both the polysomal and free (post-polysomal) RNP compartments. From each fraction the mRNP encoding histone H4 or MHC was purified by hybrid selection to a cloned human histone H4 gene or MHC cDNA. A unique set of 6-16 proteins was found bound to each of the specific mRNP complexes. These proteins were a subset of the total population of either polysomal or free RNP proteins and some proteins appeared common among the different hybrid-selected RNP fractions. The results demonstrate that (a) mRNAs bind a different set of proteins depending upon whether they are present in the polysomal or free mRNP fraction; (b) the presence of poly(A) sequences affects the proteins which bind to H4 mRNA in the free RNP compartment.

A unique subspecies of histone H4 mRNA from rat myoblasts contains poly(A).

Fractionation of rat L6 myoblast histone H4 mRNA into its three component subspecies revealed that one of the major subspecies (H4-1) contained poly(A). The unique poly(A)+ H4 mRNA makes up about 8% of the total polysomal H4 mRNA population detected. Unlike the poly(A)- histone mRNAs, whose levels are reduced by greater than 95% when myoblasts differentiate into myotubes, the poly(A)+ subspecies is reduced by only 70%. The poly(A)+ H4 mRNA from myotubes incubated with actinomycin D decays with a half-life of 37-42 min, which is similar to that obtained for the poly(A)- H4 mRNAs in myoblasts. Both the poly(A)+ and poly(A)- subspecies decay at an increased rate after inhibition of DNA synthesis. In myoblasts the poly(A)+ H4 mRNA exists almost exclusively in the polysomal compartment (greater than 95%) with little (less than 5%) in the free ribonucleoprotein (mRNA-protein or mRNP) complex compartment of the cell. Poly(A)- histone H4 mRNA subspecies, on the other hand, are distributed with approximately 80% in the polysomal compartment and 20% in the free mRNP complex compartment. The unique poly(A)+ H4 mRNA is unusual, not only in that it contains poly(A) but also in its behavior compared to poly(A)- H4 mRNAs during terminal differentiation.

Cost variances in health care: when should managers investigate?

When costs must be controlled, variance analysis can be a useful tool to implement that control. Variance analysis compares a standard of performance against actual results and investigates those differences that are felt to be the result of inefficient performance. The question becomes, which variances should be investigated? Using a decision model based on probability theory, variances can be identified that are statistically significant and require further investigation.

Solubility of calcium and zinc in model solutions based on bovine and human milks.

The solubility of calcium, magnesium, and zinc in model solutions based on the low molecular weight components of bovine and human milks was examined over a pH range similar to that found in the human digestive system. Zinc was removed from solution in all models as calcium phosphates precipitated. The pH at which precipitable calcium phosphates formed was altered by the concentration of inorganic phosphate. All calcium and zinc in a model based on human milk remain in solution up to pH 6.5 while in a model based on bovine milk they were in solution up to pH 5. The use of simple model solutions may provide information useful for understanding the different bioavailabilities of minerals from skimmed bovine and human milks.

Differentiation of rat myoblasts. Regulation of turnover of ribosomal proteins and their mRNAs.

The regulation of ribosomal proteins (r-proteins) and their mRNAs (rp-mRNAs) was studied in the L6 myoblast, a mammalian cell line which can undergo myogenesis. Upon terminal differentiation, the rate of accumulation of mature ribosomes dropped to approximately 25% of the rate found in undifferentiated myoblasts. Despite the drop in the rate of ribosome accumulation and the rate of rRNA synthesis following terminal differentiation, the rate of r-protein synthesis remained constant. The excess r-protein synthesized in myotubes was quickly degraded. The levels of rp-mRNAs were assessed before and after differentiation. Over 90% of the rp-mRNAs were found on polysomes in both myoblasts and myotubes and represented similar fractions of total poly(A)-rich mRNA. The half-lives of the rp-mRNAs averaged approximately 11 h in both myoblasts and myotubes. In vitro nuclear transcription measurements of a representative rp-mRNA (L32 mRNA) revealed that following differentiation, its rate of synthesis relative to the overall transcription rate dropped by approximately 26% in myotubes while the rate of transcription of rRNA dropped by approximately 77%. These results indicate that the coordination of r-protein and rRNA synthesis observed in myoblasts was uncoupled in myotubes at the level of transcription.

Coordinate regulation of histone mRNAs during growth and differentiation of rat myoblasts.

To determine whether histone genes are coordinately regulated, histone mRNA concentrations were measured in exponentially growing L6 myoblasts, S-phase synchronized myoblasts and in differentiating myoblasts. The levels of various histone mRNA subspecies declined rapidly and coordinately once myoblasts were given the signal to differentiate. mRNA levels were reduced on average to 1-5% of the amount observed in exponentially growing cells by 48 h after the signal to differentiate. The reductions occurred in concert with the cessation of DNA synthesis as the cells differentiated. Inhibition of DNA synthesis by treating myoblasts with Ara-C or hydroxyurea resulted in a histone mRNA half-life of 10-13 min for each of the histones examined. One example of non-coordinate regulation was observed however among the H4 mRNA subspecies in S-phase synchronized cells. The levels of two major subspecies of H4 mRNA increased coordinately in S-phase compared to levels observed in cells growing exponentially. A third subspecies of H4 mRNA on the other hand was found to decline by 50%. These studies suggest that the majority of histone mRNA subspecies are under coordinate control, although one exception has been noted among the subspecies of histone H4.