A simplified approach to stem cell mobilization in multiple myeloma patients not previously treated with alkylating agents.

High-dose chemotherapy and autologous stem cell rescue is considered a standard part of initial therapy for patients with multiple myeloma. Therefore, potential transplant candidates are generally treated with dexamethasone-based programs rather than alkylating agents to avoid stem cell toxicity. The optimal mobilizing regimen for patients with multiple myeloma has not been defined. However, aggressive chemotherapy may result in excessive morbidity and cost in this older, immunocompromised population. We retrospectively examined our experience with a well-tolerated regimen of 1.5 g/m(2) cyclophosphamide on day -10 followed by 10 microg/kg G-CSF beginning on day -7 in 50 consecutive patients with multiple myeloma and no prior alkylating agent therapy. Median stem cell collection was 4.88 x 10(6) CD34+ cells/kg per apheresis and 44 patients collected >5 x 10(6) CD34+ cells/kg within 2 days. In 36 patients, more than 10 x 10(6) CD34+ cells/kg were collected including 33 patients who required 1-2 days of collection. One patient required hospitalization for fever/neutropenia and two required weekend apheresis. We conclude that 1.5 g/m(2) cyclophosphamide plus 10 microg/kg G-CSF is a safe, effective, highly predictable mobilizing program that uniformly provided enough stem cells for one transplant and enough stem cells for two transplants.

Assessing the validity of tuberculin skin test readings by trained professionals and patients.

STUDY OBJECTIVES: To assess the validity of purified protein derivative (PPD) readings by patients and trained health-care professionals as compared with a calibrated model. DESIGN AND PARTICIPANTS: Survey of a group of patients, nurses, medical assistants, and physicians at five neighborhood health centers in the Bronx, NY. INTERVENTIONS: Participants were asked to read a calibrated model with four PPD indurations measuring 0 mm, 3 mm, 7 mm, and 13 mm. Indurations > or = 5 mm were to be considered "positive" reactions. MEASUREMENTS AND RESULTS: Data were obtained from 233 patients and 80 trained professionals. All patients correctly measured the 0-mm induration site and were able to detect the presence of an induration in 99.3% of possible observations. Compared with professionals, patients had more variability in measurements and interpretations of the 3-, 7-, and 13-mm sites. Professionals detected 100% of all indurations. Patients' specificity for the 0- and 3-mm sites was 97.4% and 62.7%, respectively; whereas sensitivity for the 7- and 13-mm sites was 68.2% and 89.3%, respectively. Professionals' specificity for the 0- and 3-mm sites was 98.7% and 65.3%, respectively; their sensitivity for the 7- and 13-mm sites was 86.7% and 97.3%, respectively. Seventy percent of professionals agreed that the model was a realistic representation of PPD indurations. CONCLUSIONS: Patients can reliably distinguish between the presence and absence of an induration at a PPD injection site. They are not as reliable in the measurement and interpretation of test reactions. Professionals had considerable variability in their assessments of PPDs but were more precise overall in their assessments than patients.

Photoaffinity labeling of rhodopsin and bacteriorhodopsin.

Photoaffinity labeling with bovine rhodopsin using a retinal with a fixed 11-cis-ene cross-linked exclusively to Trp-265/Leu-266 in helix F, showing that the beta-ionone C-3 is close to helix F. Moreover, since these labeled amino acids are in the middle of helix F, while the Schiff-base linkage to Lys-296 at the other terminus of the chromophore is also in the middle of helix G, the chromophore lies horizontally near the center of the lipid bilayer. In bacteriorhodopsin, photoaffinity studies using a retinal with a C-10 tritiated phenylazide appended through a 13 A spacer cross-linked to Arg-175/Asn-176 on the cytoplasmic side of helix F; this indicates that 9-Me points toward the extracellular space. This result agrees with our earlier studies with 9-sulfate analogs but is opposite to that deduced by biophysical measurements.

Separation of the sticky peptides from membrane proteins by high-performance liquid chromatography in a normal-phase system.

The amphiphilic peptides obtained upon cleavage of membrane proteins, including numerous receptors, are recalcitrant to most separation techniques as a consequence of their limited solubility and tendency to aggregate and adsorb to surfaces. This paper describes HPLC systems that can separate these "sticky" peptides on silica and aminopropyl-modified silica columns with a mobile phase consisting of a mixture of chloroform/methanol/isopropylamine. The protocols developed have been applied to synthetic M1 and M2 peptides, which constitute part of the transmembrane domain of glutamate-gated ion-channel proteins. Four of these M1 and M2 peptides were separated from minor synthetic impurities, and a 23-mer was baseline separated from a 28-mer. The HPLC procedures have also led to purification of the 10 peptides resulting from cyanogen bromide cleavage of bacteriorhodopsin, peptides which have so far eluded HPLC separation despite numerous attempts. These HPLC protocols have been used to purify peptides ranging from 4 to 50 amino acids in high yield while the columns continued to resolve sharp peaks after more than 100 separation runs over a 6-month period. These new HPLC systems offer an efficient method for the isolation and analysis of this important albeit troublesome class of peptides.

Ligand binding by a recombinant insect juvenile hormone binding protein.

A cDNA for the hemolymph juvenile hormone binding protein (JHBP) of larval Manduca sexta has been isolated, sequenced, and expressed in an insect cell line. A recombinant baculovirus, containing the JHBP cDNA fused to the p10 promoter of Autographa californica nuclear polyhedrosis virus, was constructed. Insect cells (Sf9) infected with this virus secreted recombinant JHBP (rJHBP) into the medium (> 50 micrograms/mL), and cotranslational removal of an 18 amino acid leader sequence was observed. rJHBP was cross-reactive with an antiserum prepared to the hemolymph JHBP and was specifically labeled by [3H]EHDA, a photoaffinity analog of JH II, demonstrating that rJHBP was an isoform of the previously reported 32-kDa JHBP [Lerro, K. A., & Prestwich, G.D. (1990) J. Biol. Chem. 265, 19800-19806]. rJHBP was purified from insect cell medium to homogeneity by ion-exchange and gel-filtration chromatography. The purified rJHBP had a higher affinity (KD = 11 nM for JH I and KD = 42 nM for JH II) than that reported for crude hemolymph JHBP (KD = 80 nM for JH I). The circular dichroism (CD) spectrum of purified rJHBP indicated 34% alpha-helix and 23% beta-sheet. The CD spectra of rJHBP in the presence and absence of JH II were the same, indicating no change in secondary structure induced by ligand binding. Thus, the rJHBP expressed in insect cells binds JHs and is suitable for structural and functional analysis.

Cell associated elastase activities of rat mammary tumour cells.

As part of our studies into the role of tumour cell proteinases in cancer invasion, we have adapted a fluorogenic assay to measure the elastase activities of intact rat mammary adenocarcinoma cells using the elastase specific substrates Cbz-Ala-Ala-Pro-Val-6-aminoquinoline and Ac-Ala-Ala-Pro-Ala-7-amino-4-methylcoumarin. This is a sensitive assay which enables rapid (30-120 min) measurement of enzyme activities under conditions of physiological pH and ionic strength and can differentiate between cell-associated and secreted enzyme activities. As the substrates are non-toxic and the method is non-invasive, cells can be reclaimed for further studies. This method thus provides a useful means for screening intact cells for elastase activity. Cell-surface elastase extracts were inhibited by phenylmethylsulphonyl fluoride but not by EDTA, indicating that they are serine proteinases. Extracts also degraded insoluble elastin confirming that these rat mammary adenocarcinoma cells produce elastase.

Cloning and sequencing of a cDNA for the hemolymph juvenile hormone binding protein of larval Manduca sexta.

A cDNA for the hemolymph juvenile hormone binding protein (JHBP) of larval Manduca sexta has been cloned and sequenced. The JHBP was purified to homogeneity from fifth instar larval hemolymph using gel filtration chromatography, ion exchange chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyclonal rabbit antibodies, generated in response to this protein, were used to identify and isolate JHBP cDNAs from a fat body expression library in bacteriophage lambda ZAPII. Eleven putative JHBP cDNA clones were isolated and subcloned into Bluescript plasmid; cDNA inserts were approximately 750 base pairs in length. A 36-kDa immunoreactive protein was expressed from these plasmids; this beta-galactosidase fusion protein, like the authentic 32-kDa JHBP, was specifically photoaffinity labeled with [3H] epoxyhomofarnesyl diazoacetate (EHDA). Single-stranded DNA from one clone was sequenced by the Sanger dideoxynucleotide method, using deletion and custom primer techniques. A mature translation product was identified which had 226 amino acid residues, a molecular mass of 25,111 daltons, and a predicted isoelectric point (pI) of 5.40. The cDNA correctly predicts the N-terminal amino acid sequence and the amino acid composition of an authentic M. sexta hemolymph JHBP. A computer search of protein and nucleic acid data bases failed to reveal any related sequences. Thus, M. sexta hemolymph JHBP appears to be the first member of a new superfamily of insect hormone binding proteins.

Polyacrylamide gel miniaturization improves protein visualization and autoradiographic detection.

Polyacrylamide gels shrink to one-quarter of their original area when soaked in a 50% (w/v) solution of polyethylene glycol. Gel miniaturization improves the contrast of protein bands, with four valuable consequences. (i) A 5- to 10-fold increase in sensitivity for Coomassie blue is observed. (ii) Gels are more durable; i.e., they resist tearing when wet and they do not crack during drying under vacuum. (iii) Shrunken gels give sharper photographic images and provide better interlane protein band comparisons. (iv) Condensed protein bands lead to an increased sensitivity for detecting low-abundance, radioactively-labeled proteins by fluorography.