Workplace substance abuse impairment: the occupational health care provider's role.

Substance abuse continues to impact the workplace. Impaired employees impact the safety of themselves, fellow employees, the public, and the physical plant. Occupational and environmental health care providers must be familiar with the seven psychoactive drug categories. In addition, they must consider other causes for behavior, such as medical, psychological, toxicological, or strictly performance issues. A standardized process should be developed and used when providing work fitness impairment evaluations.

Hsc70-binding peptides selected from a phage display peptide library that resemble organellar targeting sequences.

A 15-mer phage display random peptide library was screened with purified bovine Hsc70, and nucleotide sequence analysis of the selected clones showed a large enrichment for peptides containing basic sequences with at least KK, KR, or RR. Binding affinity for Hsc70 of representative peptides increased dramatically for heptamers compared with hexamers. The peptide NIVRKKK had the highest affinity for Hsc70, and substitution analyses showed that hydrophobic residues followed by basic residues play important roles in maintaining this affinity. In contrast, NIVRKKK was a weaker stimulator of the Hsc70 ATPase activity compared with pigeon cytochrome c peptide and FYQLALT, a peptide optimized for binding to Hsc70. FYQLALT effectively blocked the binding of NIVRKKK to Hsc70, possibly by causing a conformational change that masked Hsc70's binding site for the basic peptide. Two hypotheses are offered to explain the two different peptide motifs. First, it is proposed that Hsc70 recognizes two different amino acid sequence motifs in its dual roles of chaperoning proteins to organelles (NIVRKKK-like sequences) and facilitating protein folding (FYQLALT-like sequences). Second, the NIVRKKK motif may be used to bind certain folded proteins with which Hsc70 interacts, such as itself, p53, and Dnaj2.

BR96 sFv-PE40, a potent single-chain immunotoxin that selectively kills carcinoma cells.

We have constructed a single-chain immunotoxin composed of the carcinoma-reactive antibody BR96 and a truncated form of Pseudomonas exotoxin. The chimeric molecule, BR96 sFv-PE40, was expressed in Escherichia coli and localized to the inclusion bodies. We purified and identified two species of BR96 sFv-PE40, monomers and aggregates. The monomeric form was able to bind well to the BR96 antigen, a Lewisy-related antigen, while the aggregate was not. The binding affinity of the monomeric recombinant immunotoxin was 5-fold less than intact BR96 IgG, and its specificity for the BR96 antigen was confirmed by competition analysis. Monomeric BR96 sFv-PE40 was found to be extremely cytotoxic against cancer cells displaying the BR96 antigen. The cytotoxicity of the fusion protein correlates directly with antigen density on the tumor cell lines tested. The breast carcinoma cell line MCF-7, which has the highest density of BR96 antigen, was the most sensitive to BR96 sFv-PE40, with a concentration producing 50% protein synthesis inhibition of 5 pM. BR96 sFv-PE40 was found to have a t1/2 in serum of 28.5 min in athymic mice, compared to that of the chemical conjugate, chiBR96-LysPE40, which was 54 min. These data indicate that the single-chain immunotoxin BR96 sFv-PE40 is a potent inhibitor of protein synthesis in target cell lines and may be an effective agent for the treatment of cancer.

Expression of truncated forms of the bovine growth hormone gene in cultured mouse cells.

A synthetic oligonucleotide, 5'-d(CTAGT-CTAGACTAG)-3' which encodes translational termination codons in three reading frames, was inserted into either exon IV (pbGH-4A) or V (pbGH-5A) of the bovine growth hormone gene. The resultant plasmids, under the transcriptional regulation of the mouse metallothionein 1 promoter, were introduced into cultured mouse L-cells or rat GH3 cells. Compared to wild type bGH RNA, bGH-specific RNA transiently expressed from pBGH-5A or pBGH-4A DNA in mouse L-cells was similar or slightly smaller in size, respectively. Unexpectedly, bGH-4A RNA lacked exon IV sequences. Immunofluorescence and immunoprecipitation analyses revealed that wild type bGH was localized to the Golgi apparatus, while truncated hormones were confined to the cytoplasmic compartment of transfected cells. In addition, truncated hormones were shown to be secretion-defective albeit the bGH signal peptide was efficiently and precisely processed. Thus, structural alterations in the bGH gene can dramatically affect bGH precursor mRNA processing and hormone localization within cultured mouse fibroblast or rat pituitary cells.

Effects of a leucine analog on growth hormone processing and secretion by cultured cells.

Bovine and rat growth hormones (bGH and rGH, respectively) possess signal peptides that direct the hormone to the secretory pathway and are proteolytically cleaved prior to secretion. Previous in vitro translation studies indicated that incorporation of the polar leucine analog beta-hydroxyleucine into de novo synthesized polypeptides inhibits signal peptide function. To test the effects of this analog on GH secretion by cultured animal cells, transfections of mouse L-cells with a bGH expression plasmid or metabolic labeling of endogenous rGH in anterior pituitary cells was performed in the absence or presence of beta-hydroxyleucine. Transient expression of bGH in mouse L-cells or endogenous expression of rGH in anterior pituitary cells resulted in an accumulation of GH in the culture medium. Treatment with beta-hydroxyleucine resulted in a block in secretion as evidenced by an accumulation of GHs within these cells. Amino-terminal sequencing of the intracellular form of the analog-substituted GHs demonstrated accurate signal peptide cleavage. In contrast, in vitro translations of bGH RNA performed in the presence of beta-hydroxyleucine and microsomal membranes resulted in the inhibition of signal peptide cleavage. The results suggest that beta-hydroxyleucine can uncouple signal peptide processing and protein secretion in cultured cells.

In-vitro mutagenesis of the bovine growth hormone gene.

The biological activities of bovine growth hormone (bGH) were studied in a transgenic mouse model system. The following experimental design was used: (1) in-vitro mutagenesis of the bGH gene; (2) expression of the mutated gene in cultured mouse cells under transcriptional regulation of the mouse metallothionein I promoter; (3) binding studies of the mutated and wild-type protein to mouse liver membrane preparations; (4) generation of transgenic mice which express the mutant hormone; and (5) growth rate analysis of transgenic mice. Removal of the alanine +1 codon from the bGH gene or a substitution of serine for cysteine 189 does not affect the ability of the mutant protein to influence transgenic mouse growth. Also, mutations which increase the hydrophobicity within the bGH alpha helix 3 region (amino acid residues 109-126) do not alter the enhanced growth rate in transgenic mice which express these mutated bGH proteins.

Monoclonal antibodies reveal antigenic differences in refractile bodies of avian Eimeria sporozoites.

Monoclonal antibodies were developed against refractile body antigens of 4 species of avian Eimeria, E. meleagrimitis, E. adenoeides, E. acervulina, and E. tenella. Although antibodies from 8 different cell lines were used in this study, all produced similar fluorescent and gold-labeling patterns. By immunofluorescent antibody techniques, 5 of the 8 antibodies cross-reacted with all 4 of the Eimeria species that were examined; the other 3 antibodies reacted only with the species against which they were produced or with a limited number of species. In Western blot analyses using SDS-solubilized sporozoites as antigen, 4 of the cross-reactive antibodies recognized multiple bands; the predominant bands had molecular weights of approximately 23, 45, and 90 kilodaltons (kDa). Two of the antibodies with more limited reactivity recognized either a single band at 23 kDa (91C7), or bands at 23 and 45 kDa (4115); another reacted only with several bands greater than 100 kDa (4D10). The molecular weights of the antigens did not decrease markedly after digestion with N-glycanase F, indicating that if the refractile body antigens contained significant amounts of N-linked carbohydrate it was refractory to the enzyme. Collectively, the data indicate that antigens of the sporozoite refractile bodies differ among the Eimeria species. Some antigens are conserved, whereas others differ in distribution or frequency among the individual species.

Hybridoma antibody characterization of stage-specific and stage-cross-reactive antigens of Eimeria tenella.

Hybridoma antibodies (HAb) have been raised against the sporozoite stage of 3 species of avian coccidia. These HAb were utilized in Western blot analysis, resulting in the immunoenzymatic detection of sporozoite and merozoite antigens of 1 species, Eimeria tenella. The 5 HAb specific for the sporozoite stage showed either single bands at 22 and 28 kDa or a large diffuse band in the 7-10-kDa range. The 4 HAb that cross-reacted with both asexual stages recognized either a single sporozoite or merozoite antigen of 90 kDa, or multiple antigens (47-69 kDa) for both stages. The 9 HAb demonstrated 5 different immunofluorescent antibody (IFA) patterns, and the 4 cross-reactive HAb showed similar IFA patterns with both asexual stages of E. tenella. The sporozoite-specific HAb which identified the 22, 7-10, and 7-8 kDa antigens showed surface, surface-internal, or internal IFA patterns. The other sporozoite-specific HAb, which labeled the 28-kDa antigen, stained the refractile body. The IFA of the 4 stage-cross-reactive HAb, which recognized the 45-60-kDa and the 90- or 47-69-kDa antigens, localized these antigens to the surface and tip, respectively. Rabbit anti-sporozoite serum appeared to recognize all of the sporozoite and merozoite antigens identified by the HAb as well as a variety of additional stage-cross-reactive antigens.

Eukaryotic ribosomes can recognize preproinsulin initiation codons irrespective of their position relative to the 5' end of mRNA.

The functional assay of a eukaryotic mRNA, into which additional AUG codons have been introduced by in vitro mutagenesis, shows that a translational initiation site need not necessarily be the nearest AUG codon to the 5' end of a mRNA. Hence, the sequence surrounding the AUG, and not simply its position relative to the 5' end of mRNA, appears to be important in determining initiation efficiency.

The molecular characterization of three transcriptional mutations in the yeast iso-2-cytochrome c gene.

Three mutations, each of which causes overproduction of iso-2-cytochrome c, were characterized biochemically. Two, CYP3-4 and CYP3-15, were previously shown to be cis-dominant and map to the CYC7 locus which encodes the iso-2 protein, while the third, cyp1-16, maps to an unliked locus. All three mutations caused dramatically increased levels of transcription of the CYC7 gene, and the CYC7 mRNA in mutant cells was found to be the same size as that in wild type cells. The CYP3-4 mutation was found to be caused by the integration of a transposable element, Tyl, 269 base pairs 5' to the coding sequences. The CYP3-15 mutation was also found to alter the DNA, probably through a deletion or inversion with one endpoint 285 base pairs upstream from the coding sequence. The CYC7 gene in both wild type and mutant cells was not subject to catabolite repression.