Cryptococcosis in Gilbert's and long-nosed potoroo.

Two cases of fatal cryptococcosis are described, one of Cryptococcus neoformans infection in a Gilbert's potoroo (Potorous gilbertii) and one of Cryptococcus gattii infection in a long-nosed potoroo (Potorous tridactylus). The diagnoses were confirmed by culture and specific immunohistochemistry, respectively. The long-nosed potoroo tested positive using the latex cryptococcal antigen test (LCAT), whereas the Gilbert's potoroo had a negative LCAT result despite having advanced disease of some duration. In both cases, the clinical presentation was a progressive neurologic disease associated with a central nervous system infection. Pulmonary infection was also observed in the long-nosed potoroo. Specific treatment with antifungal agents was unsuccessful in the long-nosed potoroo.

Efficient chromosomal mapping of a methylcholanthrene-induced tumor antigen by CTL immunoselection.

It has been difficult to genetically map the genes encoding tumor Ags because they arise as a consequence of somatic mutational events. CTL-mediated immunoselection can impose potent immunoselective pressure against tumor cells, resulting in the survival of rare tumor Ag-loss variants. We subjected a heterozygous 3-methylcholanthrene-induced murine sarcoma cell line to CTL immunoselection, selecting for the loss of a tumor-specific Ag, recognized antigen from MCA-induced tumor 1 (Ram1). Several variants eluded CTL recognition by genetic loss of the hemizygously expressed tumor-specific Ag epitope. A frequently observed genetic escape mechanism was spontaneous mitotic recombination resulting in loss of heterozygosity on chromosome 4. Higher density genetic analyses along with functional confirmation with an independently produced chromosome 4 loss of heterozygosity variant positioned the Ram1 locus to a distal 7.1 cM interval on chromosome 4. This region of the mouse genome is rich in tumor-modifier genes and this positioning of Ram1 may thus provide insight into the genetic basis of 3-methycholanthrene-induced tumor Ags.

Biochemical and immunogenetic analysis of an immunodominant peptide (B6dom1) encoded by the classical H7 minor histocompatibility locus.

Of the many minor histocompatibility (H) Ags that have been detected in mice, the ability to induce graft vs host disease (GVHD) after bone marrow transplantation is restricted to a limited number of immunodominant Ags. One such murine Ag, B6dom1, is presented by the H2-Db MHC class I molecule. We present biochemical evidence that the natural B6dom1 peptide is indistinguishable from AAPDNRETF, and we show that this peptide can be isolated from a wide array of tissues, with highest levels from the lymphoid organs and lung. Moreover, we employ a novel, somatic cell selection technique involving CTL-mediated immunoselection coupled with classical genetics, to show that B6dom1 is encoded by the H7 minor H locus originally discovered approximately 40 years ago. These studies provide a molecular genetic framework for understanding B6dom1, and exemplify the fact that mouse minor H loci that encode immunodominant CTL epitopes can correspond to classical H loci originally identified by their ability to confer strong resistance to tumor transplantation. Additionally, these studies demonstrate the utility of somatic cell selection approaches toward resolving H Ag immunogenetics.

The molecular and functional characterization of a dominant minor H antigen, H60.

Minor histocompatibility (H) Ags elicit T cell responses and thereby cause chronic graft rejection and graft-vs-host disease among MHC identical individuals. Although numerous independent H loci exist in mice of a given MHC haplotype, certain H Ags dominate the immune response and are thus of considerable conceptual and therapeutic importance. To identify these H Ags and their genes, lacZ-inducible CD8+ T cell hybrids were generated by immunizing C57BL/6 (B6) mice with MHC identical BALB.B spleen cells. The cDNA clones encoding the precursor for the antigenic peptide/Kb MHC class I complex were isolated by expression cloning using the BCZ39.84 T cell as a probe. The cDNAs defined a new H locus (termed H60), located on mouse chromosome 10, and encoded a novel protein that contains the naturally processed octapeptide LTFNYRNL (LYL8) presented by the Kb MHC molecule. Southern blot analysis revealed that the H60 locus was polymorphic among the BALB and the B6 strains. However, none of the H60 transcripts expressed in the donor BALB spleen were detected in the host B6 strain. The expression and immunogenicity of the LYL8/Kb complex in BALB.B and CXB recombinant inbred strains strongly suggested that the H60 locus may account for one of the previously described antigenic activity among these strains. The results establish the source of an immunodominant autosomal minor H Ag that, by its differential transcription in the donor vs the host strains, provides a novel peptide/MHC target for host CD8+ T cells.

In situ hybridization for gastrin-releasing peptide receptor (GRP receptor) expression in prostatic carcinoma.

Bombesin-like peptides (BLPs), which have been implicated in the regulation of growth of prostatic carcinoma cells, are a product of neuroendocrine cells frequently found in prostate tissue and are postulated to play a role in the initiation or progression of prostatic carcinoma. In this report, we examined the expression, in human prostate tissue, of mRNA encoding the 3 known receptors that respond to BLPs in humans, i.e., gastrin-releasing peptide (GRP) receptor, neuromedin B (NMB) receptor and bombesin receptor subtype 3 (BRS-3). Competitive rt-PCR experiments demonstrated the widespread but variable expression of GRP receptor mRNA in fresh-frozen specimens of prostatic carcinoma (12 cases) and benign prostatic hypertrophy (6 cases). NMB receptor mRNA expression was also widespread, but its level was less variable than GRP receptor message. In contrast, we could not detect BRS-3 mRNA in most tissue samples by rt-PCR. To address which cells in the prostate express the GRP receptor, we used in situ hybridization methods to stain selectively GRP receptor mRNA. GRP receptor mRNA was expressed predominantly in the luminal and basal epithelial cells in both histologically normal and cancerous glands within sections of normal (3 cases) and diseased (37 cases) tissue. GRP receptor mRNA staining in cancerous tissue ranged widely from very intense to not detectable (about 30% of the cases), while normal tissue consistently displayed a low level of message staining. Taken together, our results demonstrate expression of the GRP receptor in a high percentage of basal and/or luminal epithelial cells of normal and diseased prostate tissues.

Proliferative response of human and animal tumours to surgical wounding of normal tissues: onset, duration and inhibition.

Acceleration of secondary tumour growth and metastases following excision of a primary tumour has been attributed to the consequent removal of primary tumour-generated inhibitory factors. However, our studies have shown that surgical wounding of normal tissues significantly stimulated the growth of malignant tissues without the concomitant presence or excision of a tumour mass. A humoral stimulating component was indicated by the proliferative response of tumours and metastases distant from the surgical wound. All 16 human and murine tumours, of nine different histologies, showed a measurable acceleration of growth when implanted in surgically treated animals, suggesting that the ability of malignant tissue to respond to surgical wounding of normal tissue was not histologically or species specific. The proliferative surge of malignant tissues was detectable soon after wounding and had a duration of 2-3 days. The surgical wound as the source of the tumour-stimulating factor(s) was affirmed by the significant inhibition of tumour proliferative responses when a somatostatin analogue was applied topically to the surgical wound within 1 h of wounding, and/or during the critical tumour-stimulatory period of 1-2 days after wounding. A potential therapeutic window for reducing a risk factor that may be inadvertently imposed upon every surgical/oncology patient is indicated.

Detection of somatostatin receptor subtype 2 (SSTR2) in established tumors and tumor cell lines: evidence for SSTR2 heterogeneity.

The somatostatin receptor subtype 2 (SSTR2) was detected in a wide range of human and rat tumors using in vitro receptor binding ([125I]MK-678), receptor gene expression analysis, and immunoblotting techniques. The highest receptor concentrations were observed in the rat AR42J pancreatic and human small cell lung cancer (SCLC) cell lines, NCI-H69 and NCI-H345, with much lower levels detected in breast, prostate, melanoma, and hepatic tumors. Several human pancreas tumors were devoid of SSTR2. For all tumors showing detectable [125I]MK-678 binding, SSTR2 receptor mRNA was expressed. Furthermore, a mRNA transcript corresponding to a truncated isoform of SSTR2 was detected at low levels in the human SCLC NCI-H69 cell line, and likely represents a human homologue of rodent SSTR2B. Immunoblotting analysis using the SSTR2-specific antibody, 2e3, detected multiple immunoreactive protein species, including a predominant 150-kDa molecule, which could be blocked by the SSTR2-derived 2e3 peptide. Somatostatin (SRIF) peptides with high SSTR2 affinity and antiproliferative properties were potent inhibitors of [125I]MK-678 binding to several tumor types, suggesting that they may exert antitumor effects via the SSTR2 receptor.

Brain protein kinase PK40erk converts TAU into a PHF-like form as found in Alzheimer's disease.

The novel protein kinase PK40 (1) was characterized by its ability to phosphorylate Lys-Ser-Pro sites in neurofilament and TAU proteins. PK40 is now recognized to be a member of the family of External-stimulus Regulated Kinases (ERKs) by its reactivity with ERK-specific antibodies and will therefore be called PK40erk. Bovine TAU or recombinant human TAU proteins can be hyperphosphorylated by PK40erk to produce the electrophoretic mobility shifts and certain immunochemical properties characteristic of PHF-TAU isolated from Alzheimer's disease brain tissue. PK40erk may play a crucial role in the etiology of this disease.

Somatostatin receptor subtype gene expression in human and rodent tumors.

Somatostatin (SRIF) analogues display anti-tumor properties believed to be mediated by specific cell surface somatostatin receptors (SSTR). SSTR subtypes have unique pharmacological properties, including specific GTP-binding protein coupling, ion channel regulation, and cAMP inhibition; therefore, identification of isotypes expressed in tumor cells facilitates current efforts to design potent anti-tumor SRIF analogues. Human and rodent solid, transplantable tumors and tumor cell lines were examined for gene expression of SSTR1, SSTR2 and SSTR3 by reverse transcription of tumor mRNA and subsequent amplification of cDNA by the polymerase chain reaction, using SSTR subtype-specific oligonucleotide primers. SSTR2 mRNA transcripts were observed in all of the tumor cell lines examined. SSTR1 gene expression was seen in several human and rat tumor types, and SSTR3 gene expression observed in two rodent tumor types. SSTR mRNA-positive tumors are expected to possess membrane-bound receptors which could potentially interact with anti-tumor SRIF analogues.

Phylogenetic analysis of Aquaspirillum magnetotacticum using polymerase chain reaction-amplified 16S rRNA-specific DNA.

The 16S rRNA gene of the magnetotactic magnetogen Aquaspirillum magnetotacticum MS1 was amplified by a polymerase chain reaction, using two eubacterial consensus oligodeoxynucleotide primers flanking the majority of the 16S rRNA gene, cloned, and sequenced. Phylogenetic analysis revealed that A. magnetotacticum MS1 belongs to the alpha-group of proteobacteria. This assignment offers perspective on the biochemical properties of A. magnetotacticum, since this organism is expected to have the general properties that are common to this phylogenetic group.

Electroporation and conjugal plasmid transfer to members of the genus Aquaspirillum.

Electroporation methods and conjugal matings were used to transfer several plasmid vectors to Aquaspirillum dispar and Aquaspirillum itersonii. The incompatibility P class plasmid RP4 was conjugally transferred from Escherichia coli HB101 to these spirilla, and the transconjugants subsequently donated the molecule to plasmid-free E. coli and A. dispar strains via conjugal matings. High-voltage electrotransformation was used to transfer plasmids pUCD2, pSa151 and RP4 to A. dispar and A. itersonii, at efficiencies as high as 3 x 10(4) transformants per micrograms plasmid DNA. RP4 DNA isolated from spirillum hosts, but not RP4 from E. coli cells was successfully transferred to A. dispar and A. itersonii by electrotransformation, suggesting that modification and/or restriction activity may be present in these Aquaspirillum species.