The sequence of the cloned Bacillus amyloliquefaciens subtilisin gene suggested that this secreted serine protease is produced as a larger precursor, designated preprosubtilisin [Wells,

The Lactobacillus bulgaricus Il-galactosidase gene was cloned on a ca. 7-kilobase-pair HindIII fragment in the vector pKK223-3 and expressed in Escherichia coli by using its own promoter. The nucleotide sequence of the gene and approximately 400 bases of 3'-and 5'-flanking sequences was determined. The amino acid sequence of the 6-galactosidase, deduced from the nucleotide sequence of the gene, yielded a monomeric molecular mass of ca. 114 kilodaltons, slightly smaller than the E. coli lacZ and Klebsiella pneumoniae lacZ enzymes but larger than the E. coli evolved (ebgA) 0-galactosidase. The cloned P-galactosidase was found to be indistinguishable from the native enzyme by several criteria. From amino acid sequence alignments, the L. bulgaricus , -galactosidase has a 30 to 34% similarity to the E. coli lacZ, E. coli ebgA, and K. pneumoniae lacZ enzymes. There are seven regions of high similarity common to all four of these PI-galactosidases. Also, the putative active-site residues (Glu-461 and Tyr-503 in the E. coli lacZ ,I-galactosidase) are conserved in the L. bulgaricus enzyme as well as in the other two ,I-galactosidases mentioned above. The conservation of active-site amino acids and the large regions of similarity suggest that all four of these PI-galactosidases evolved from a common ancestral gene. However, these enzymes are quite different from the thermophilic 0-galactosidase encoded by the Bacillus stearothermophilus bgaB gene.Little is known about the specific amino acids involved in the substrate binding and catalysis of the Escherichia coli lacZ P-galactosidase, even though this enzyme has been studied for many years (35). From iodination (8, 15), fluorotyrosine substitution (27), and active-site-directed inhibitor (11,12,23) experiments, Tyr-503 is thought to be the proton-donating species needed for catalysis. Primarily by the use of active-site-directed reagents (11,13)

Experiments in animal models suggest that it is feasible to consider hepatic gene therapy using a strategy in which hepatocytes would be isolated by partial hepatectomy, transduced with recombinant retroviral vectors contalning genes of therapeutic importance, and then transplanted back into the patient by autologous hepatocellular transplantation. The application of this strategy in clinical trials will require adapting these methods to human cells. We describe the transduction of primary human hepatocytes with two forms of retroviral vectors: amphotropic vectors, which have been used previously in clinical trials, and xenotropic vectors, which have a different host range. Human hepatocytes were harvested from organs preserved in Belzer's solution and were cultivated in a serum-free, tyrosine-free, hormonally defined medium. These cells proliferated for 3-5 days in culture, exhibited characteristic hepatocyte morphology, and expressed liverspecific functions, including phenylalanine hydroxylase, a,-antitrypsin, and glutamine synthase. Transduction with an amphotropic LNL6 retroviral vector resulted in stable incorporation of the provirus into 1% of the cells as estimated by semiquantitative PCR. Consistently higher transduction efficiencies (as much as 10% of the cells) were observed with a xenotropic N2 vector. These data support the feasibility of using LNL6 as a marker gene in clinical trials of hepatocellular transplantation. These data also suggest that the efficiency of transducing hepatocytes with amphotropic vectors in animal models may not accurately reflect the utility of these vectors for human applications. Consideration should be given to the use of xenotropic vectors for optimizing the efficiency of transduction for human applications.

Despite reports of successful cryopreservation of primary human hepatocytes, existing methods do not produce sufficient recovery of viable cells to meet the needs of basic research or clinical trials of hepatocellular transplantation. We now describe a protocol for efficient cryopreservation of primary human hepatocytes using University of Wisconsin (UW) solution, fetal bovine serum, and dimethyl sulfoxide (DMSO). This method provides > 90% viability of differentiated, primary human hepatocytes 8 mo after cryopreservation as measured by trypan blue exclusion, preserves hepatocyte morphology, liver-specific gene expression (alpha 1 antitrypsin), and replication. The effectiveness of UW solution as a cryopreservative agent suggests that metabolic as well as ultrastructural factors may be important in the effective cryopreservation of primary human hepatocytes. The present method represents an effective protocol for cryopreserving differentiated primary human hepatocytes for research. This method may allow characterization and banking of human hepatocytes for clinical applications, including hepatocellular transplantation and hepatic assist devices.

IntroductionTalaromyces marneffei infection is a systemic mycosis, caused by a dimorphic fungus, an opportunistic pathogen formerly known as Penicillium marneffei. This disease is endemic to Southeast Asia and common in human immunodeficiency virus (HIV) infected patients with low CD4 counts. Here we present a very rarely reported case of Talaromyces marneffei infection in an apparent non-immunosuppressed patient presenting decades later in a non-endemic setting (United States).Presentation of caseOur patient was a 75-year-old Caucasian Navy veteran, who served in Vietnam as a part of the Swift Boat service in 1966. He presented to his primary care provider with uncontrolled nonproductive cough and abnormal chest computerized tomography. Bronchoscopy specimens showed Talaromyces. He was empirically treated with itraconazole and then switched to voriconazole after confirmation of diagnosis but he later deteriorated was changed to liposomal amphotericin B and isavuconazole. Patient did well for the next 90 days on isavuconazole until the therapy was stopped. Soon after stopping the medication (isavuconazole) his symptoms recurred and ultimately patient expired.DiscussionTalaromycosis generally presents as pulmonary infection with manifestations similar with other endemic fungi. It is often seen HIV patients with travel to South east Asia. Very rarely this infection is seen and reported in non-immunosuppressed and in non-endemic areas. To date there are 4 well-documented cases among non-HIV, non-endemic population.ConclusionTalaromyces can cause infection in non-HIV and non-endemic population and could be an underrecognized cause of pulmonary infections among veterans with even a remote history of exposure to the organism during deployment.