Psp and Smgb: a model for developmental and functional regulation in the rat major salivary glands.

This paper summarizes past work detailing the developmental expression, cell and organ localization and biochemical features of the proteins parotid secretory protein (PSP) and isoforms of submandibular gland protein B (SMGB), and describes the molecular characterization of the genes that encode them, Psp and Smgb. These genes appear to be related to the BPI (bactericidal/permeability-increasing protein)/LBP (lipopolysaccharide-binding protein)/PLUNC (palate, lung and nasal epithelial clone) gene family found in the oral and respiratory organs of humans, rodents and cattle. We have emphasized the diverse patterns of expression of these genes among the submandibular, sublingual and parotid salivary glands of the rat, and their potential usefulness in defining and identifying genomic regulatory mechanisms of salivary development. While Psp is expressed similarly in the mouse, the putative Smgb gene of the mouse seems not to be expressed, apparently due to the insertion, between exons 1 and 2, of a gene for a retroviral protein.

Development of the rat sublingual gland: a light and electron microscopic immunocytochemical study.

Cell differentiation in the rat sublingual gland occurs rapidly and is largely complete by birth. To study differentiation of the serous and mucous cells of the sublingual gland, we used antibodies to the secretory proteins CSP-1, SMGB, PSP, and SMGD, and sublingual mucin as specific cell markers. Glands from rats at ages 18, 19, and 20 days in utero, and postnatal days 0, 1, 5, 9, 14, 18, 25, 40, and 60 were fixed and prepared for morphological analysis and immunocytochemical labeling. At age 18 days in utero, a few cells in the developing terminal bulbs contained mucous-like apical granules that labeled with anti-mucin. Other cells had mixed granules with a peripheral lucent region and a dense core of variable size that occasionally labeled with anti-SMGD. Additionally, presumptive serous cells with small dense granules that contained CSP-1 and SMGB were present. At age 19 days in utero, the dense granules of these cells also labeled with anti-SMGD. By age 20 days in utero, mucous cells were filled with large, pale granules that labeled with anti-mucin, and serous cells had numerous dense granules containing CSP-1, SMGB, PSP, and SMGD. Fewer cells with mixed granules were seen, but dense regions present in some mucous granules (MGs) labeled with anti-SMGD. After birth, fewer MGs had dense regions, and serous cells were organized into well-formed demilunes. Except for PSP, which was undetectable after the fifth postnatal day, the pattern of immunoreactivity observed in glands of neonatal and adult animals was similar to that seen by age 20 days in utero. These results suggest that mucous and serous cells have separate developmental origins, mucous cells differentiate earlier than serous cells, and cells with mixed granules may become mucous cells.

Comparison of oral fluconazole and itraconazole for progressive, nonmeningeal coccidioidomycosis. A randomized, double-blind trial. Mycoses Study Group.

BACKGROUND: In previous open-label noncomparative clinical trials, both fluconazole and itraconazole were effective therapy for progressive forms of coccidioidomycosis. OBJECTIVE: To determine whether fluconazole or itraconazole is superior for treatment of nonmeningeal progressive coccidioidal infections. DESIGN: Randomized, double-blind, placebo-controlled trial. SETTING: 7 treatment centers in California, Arizona, and Texas. PATIENTS: 198 patients with chronic pulmonary, soft tissue, or skeletal coccidioidal infections. INTERVENTION: Oral fluconazole, 400 mg/d, or itraconazole, 200 mg twice daily. MEASUREMENTS: After 4, 8, and 12 months, a predefined scoring system was used to assess severity of infection. Findings were compared with those at baseline. RESULTS: Overall, 50% of patients (47 of 94) and 63% of patients (61 of 97) responded to 8 months of treatment with fluconazole and itraconazole, respectively (difference, 13 percentage points [95% CI, -2 to 28 percentage points]; P = 0.08). Patients with skeletal infections responded twice as frequently to itraconazole as to fluconazole. By 12 months, 57% of patients had responded to fluconazole and 72% had responded to itraconazole (difference, 15 percentage points [CI, 0.003 to 30 percentage points]; P = 0.05). Soft tissue disease was associated with increased likelihood of response, as in previous studies. Azole drug was detected in serum specimens from all but 3 patients; however, drug concentrations were not helpful in predicting outcome. Relapse rates after discontinuation of therapy did not differ significantly between groups (28% after fluconazole treatment and 18% after itraconazole treatment). Both drugs were well tolerated. CONCLUSIONS: Neither fluconazole nor itraconazole showed statistically superior efficacy in nonmeningeal coccidioidomycosis, although there is a trend toward slightly greater efficacy with itraconazole at the doses studied.

Role of IL-10 in invasive aspergillosis: increased resistance of IL-10 gene knockout mice to lethal systemic aspergillosis.

IL-10 is associated with a Th2 response, down-regulation of a Th1 response and macrophage activation. We assessed the role of IL-10 during systemic infection with Aspergillus fumigatus. Systemic aspergillosis was established in female C56B1/6 IL-10(-/-) (KO) and wild-type (WT) C57B1/6 mice by i.v. administration of 1 x 10(5)-6 x 10(5) conidia of A. fumigatus. In two experiments, KO survived longer than did WT (P < 0.001). Determination of fungal burdens in the kidneys and brain showed that KO carried significantly lower burdens in both organs than did WT on day 3 (P < 0.001). Semiquantitative histological analyses showed fewer inflammatory foci/mm2 in brain and kidneys of KO than WT (P < 0.03 and < 0.001, respectively) and that extent of infection and associated tissue injury were greater in WT. Although beneficial in some bacterial infections, exogenous IL-10 has been shown deleterious in models of fungal infection. Our data indicate IL-10 is deleterious during systemic aspergillosis infection, increasing the host susceptibility to lethal infection. We speculate this might be related to greater Th2 or lesser Th1 responses, or down-regulation of macrophage responses, in WT compared with KO.

Submandibular gland adenocarcinoma of intercalated duct origin in Smgb-Tag mice.

A line of transgenic mice that develops submandibular gland adenocarcinoma of intercalated duct origin was established. In these mice, the oncogene SV40 T antigen (Tag) is expressed from the neonatal submandibular gland secretory protein b (Smgb) gene promoter. This hybrid gene directs expression of the oncoprotein to neonatal submandibular gland proacinar and terminal tubule cells and to intercalated ducts of the adult gland. Transgene expression resulted in duct luminal cell hyperplasia as early as 20 to 30 days postnatally, which progressed to dysplasia by 3 to 4 months of age. Marked dysplasia and in situ carcinoma were evident at 4 to 6 months of age. All histologic changes were more pronounced in males. Submandibular gland adenocarcinoma developed stochastically in more than half of the adult male mice by 12 months of age (average age: 10.8 months, range: 6 to 13.5 months). Tag expression persisted in in situ carcinoma and all tumors. Using a combination of immunocytochemical and ultrastructural criteria, submandibular gland dysplasia and tumors were found to originate from intercalated ducts. The dysplastic ducts and adenocarcinoma in Smgb-Tag mice were morphologically similar to previously reported Tag-induced dysplasias of striated ducts and granular convoluted tubules and a Tag-induced adenocarcinoma of striated duct origin. These findings demonstrate that salivary gland dysplasias and tumors of similar histologic appearance can arise from distinct differentiated cell types. Analysis of the molecular changes accompanying tumor formation in Smgb-Tag mice could increase knowledge of human salivary gland tumorigenesis.

Hyphal forms in the central nervous system of patients with coccidioidomycosis.

Coccidioides immitis is a dimorphic fungus that grows as a filamentous mold in soil and as a spherule at human body temperature. The hyphal or soil form is found rarely in human tissue. We report 5 cases of coccidioidomycosis in which hyphae were found in brain tissue or spinal fluid. The presence of central nervous system plastic devices appears to be associated with morphological reversion to the saprophytic form. This reversion has implications for diagnosis and therapy and may increase the risk of obstruction of the device(s).

Structure and chromosomal localization of the rat salivary Psp and Smgb genes.

SMGB and PSP are among the most abundant products of the immature acinar cells in developing rat parotid and submandibular glands and are also products of the sublingual gland serous demilunes. Previous analysis of Smgb and Psp cDNA clones demonstrated a high degree of sequence similarity between the signal peptide-encoding and 3' untranslated regions of these transcripts, although the secreted proteins themselves are more divergent. The current study reports the upstream sequences, genomic organization and localization of the Psp and Smgb genes. Both structural genes contain nine exons and are present at 3q41-3q42, where they are arranged in tandem and separated by 21kb. In addition to the previously observed sequence similarity, Psp and Smgb are highly homologous throughout exon 1 and at 365 of 600bp immediately upstream of the transcription start site. These findings indicate that the Psp and Smgb genes arose by tandem duplication and divergence. The similar neonatal submandibular and parotid gland expression patterns observed for these genes are likely to be due to closely conserved or shared enhancer(s).

Secretory protein expression patterns during rat parotid gland development.

The rat parotid gland produces a number of well-characterized secretory proteins. Relatively little is known, however, about the onset of their synthesis and cellular localization during gland development. Secretory protein expression was studied in parotid glands of fetal and postnatal rats using light and electron microscopic immunocytochemistry and Northern blotting. Amylase, parotid secretory protein (PSP), common salivary protein-1 (CSP-1), and SMGB were first detected by immunofluorescence in parotid glands of 18 day fetuses. By 5 days after birth, light and electron microscopic immunolabeling localized all of these proteins to the secretory granules of developing acinar cells. Labeling of acinar cells for DNAse I, however, was not observed until 18 days after birth. Between 9 and 25 days, CSP-1 and SMGB reactivity of acinar cells declined, but increased in intercalated duct cells. After 25 days, CSP-1 and SMGB were found only in intercalated ducts, and amylase, PSP, and DNAse I were restricted to acinar cells. Levels of CSP-1 and SMGB mRNA were relatively constant through 21 postnatal days, but declined significantly after that. Amylase and PSP mRNA increased rapidly and continuously from five days after birth to the adult stage. In contrast, DNAse I mRNA was not detectable until 18 days after birth. The immunocytochemical and molecular analyses define three basic patterns of protein expression in the rat parotid gland: proteins whose synthesis is initiated early in development and is maintained in the acinar cells, such as amylase and PSP; proteins that are initially synthesized by immature acinar cells but are restricted to intercalated ducts in the adult gland, such as CSP-1 and SMGB; and proteins that are synthesized only by mature acinar cells and first appear during the third postnatal week, such as DNAse I. The parotid gland exhibits four distinct developmental stages: prenatal, from initiation of the gland rudiment until birth; neonatal, from 1 day up to about 9 days postnatal; transitional, from 9 days to 25 days of age; and adult, from 25 days on. Although differences exist in timing and in the specific proteins expressed, these developmental stages are similar to those seen in the rat submandibular gland. Additionally, the results support the suggestion that intercalated ducts may differentiate from the neonatal acini.

Expression of gross cystic disease fluid protein-15/Prolactin-inducible protein in rat salivary glands.

Gross cystic disease fluid protein-15 (GCDFP-15)/prolactin-inducible protein (PIP) is present at moderate levels in human submandibular and sublingual glands and is barely detectable in human parotid gland. The rodent homologue, PIP, has previously been identified in adult submandibular and lacrimal glands. Here we present the molecular characterization of rat PIP and show that this protein is a product of neonatal and adult rat submandibular, sublingual, and parotid glands. cDNA clones encoding rat PIP were isolated and sequenced. The deduced amino acid sequence of rat PIP shows 56% overall identity and 80% similarity with mouse PIP. By SDS-PAGE, secreted rat PIP has an apparent Mr of 17,000, with a minor proportion present as Mr 20-22,000 N-glycosylated forms. PIP was localized in rat salivary glands by immunogold silver staining. PIP was identified in acinar cells of developing and mature submandibular and parotid glands and at very low levels in sublingual gland serous demilunes. Typically, rat submandibular gland secretory proteins are produced by either acinar cell progenitors (Type III cells) or mature acinar cells. The expression pattern observed for PIP is similar to that previously reported for salivary peroxidase, an important component of nonimmune mucosal defense.

Characterization of the rat salivary-gland B1-immunoreactive proteins.

The B1-immunoreactive proteins (B1-IPs) are major secretory products of rat submandibular gland acinar-cell progenitors, and are also produced by neonatal and adult rat sublingual and parotid glands. In order to characterize the B1-IPs, we have previously isolated cDNA clones encoding rat parotid secretory protein (PSP; the predominant parotid B1-IP) and the related clone ZZ3, which is developmentally regulated in the neonatal submandibular gland. The remainder of the B1-IPs were uncharacterized. This report demonstrates that all of the B1-IPs are derived from the PSP and ZZ3 transcripts. Molecular cloning and Western-blot analyses using PSP- and ZZ3-specific antisera show that, of the B1-IPs, only PSP and neonatal submandibular gland protein A (SMGA) are products of the Psp gene. This finding corrects our previous assertion that SMGA is derived from ZZ3. Neonatal submandibular gland proteins B1 and B2, as well as apparent Mr 26000-28000 and Mr 18000-20000 forms in submandibular, sublingual and parotid glands, are derived from the gene encoding ZZ3 by differential N-glycosylation and by proteolytic cleavage. The apparent Mr 18000-20000 proteolytic products are significant in secretion product collected in vitro, but rare in gland homogenate and submandibular/sublingual saliva. The gene encoding ZZ3 has been named Smgb. Psp and Smgb are regulated similarly in the developing submandibular gland, but differently in the sublingual and parotid glands. The expression pattern of Psp is conserved between rat and mouse. However, no evidence for proteins derived from an Smgb-like gene was observed in neonatal mouse submandibular or sublingual glands.

Immunocytochemical studies of cell differentiation during rat salivary gland development.

The development of the rat submandibular and parotid glands has been studied using antibodies to secretory proteins as cell-specific markers. Although the morphology of the glands and the timing of the main steps of cytodifferentiation are substantially different, they have several proteins and developmental features in common. The latter include the initial formation of perinatal acini which undergo a transition to adult acini expressing a different complement of secretory proteins; the development of adult intercalated ducts (ID) from the perinatal acinar cells; and the retention of the perinatal phenotype in some cells of the ID.

Mouse submandibular gland salivary apomucin contains repeated N-glycosylation sites.

A cDNA clone encoding mouse submandibular gland salivary mucin apoprotein was isolated and characterized. The mucin cDNA encodes a protein of 273 amino acids with a calculated molecular weight of 29,606. This apomucin is approximately 60% Thr, Ser, and Pro, and has a pI of 11.25. In addition to the signal sequence, the apomucin can be divided into four structural domains. The first of these contains over 30% Thr, Ser, and Pro, but only a few probable O-glycosylation sites. The second domain contains 10 repeats, each 9 or 13 amino acids in length, with Thr representing more than 50% of the amino acids, while Ser accounts for only 2%. Each repeat begins with a putative N-glycosylation site; hence this domain likely contains both N- and O-linked oligosaccharides. The third domain lacks a repeat motif, but is rich in both Thr and Ser, and therefore is potentially highly O-glycosylated. The final domain is composed mainly of basic and non-polar amino acids and does not contain Cys. This mucin shows considerable homology with the rat submandibular salivary mucin, but little overall homology with other mucins. In situ hybridization verifies that the mucin transcript is localized primarily in the acinar cells of the submandibular gland.

Determinants of carboxyl-terminal domain translocation during prion protein biogenesis.

The prion protein (PrP) displays some unusual features in its biogenesis. In cell-free systems it can be synthesized as either an integral transmembrane protein spanning the membrane twice, with both amino and carboxyl domains in the lumen of the endoplasmic reticulum, or as a fully translocated polypeptide. A charged, extracytoplasmic region, termed the Stop Transfer Effector (STE) sequence, has been shown to direct the nascent translocating chain to stop at the adjoining hydrophobic domain to generate the first membrane-spanning region (TM1). However, the determinants of the second translocation event in the biogenesis of the transmembrane form have not been identified previously. Moreover, the relationship of transmembrane and fully translocated forms of PrP has not been well understood. Here, we report progress in resolving both of these issues. Using protein chimeras in cell-free translation systems and Xenopus oocytes, we identify the sequence which directs nascent PrP to span the membrane a second time, with its carboxyl-terminal domain in the endoplasmic reticulum lumen. Surprisingly, PrP carboxyl-terminal domain translocation does not appear to be directed by an internal signal or signal-anchor sequence located downstream of TM1, as would have been expected from studies of other multispanning membrane proteins. Rather, carboxyl-terminal domain translocation appears to be another consequence of the action of STE-TM1, that is, the same sequence responsible for generating the first membrane-spanning region. Studies of an STE-TM1-containing protein chimera in Xenopus oocytes demonstrate that most of these chains upon completion of their translation, initially span the membrane twice, with a topology similar to that of transmembrane PrP, but are carbonate-extractable. These chains have the transmembrane orientation only transiently and chase into a fully translocated form. These results support a model in which a metastable "transmembrane" intermediate, residing within the aqueous environment of the translocation channel, can be converted into either the integrated transmembrane or the fully translocated form of PrP, perhaps directed by trans-acting factor (s). Such a model may explain why stable the transmembrane isoform of PrP has not been observed in normal cells and how nascent PrP might be directed to alternate pathways of folding.

Characterization of common salivary protein 1, a product of rat submandibular, sublingual, and parotid glands.

We have isolated and characterized cDNA clones derived from a developmentally regulated neonatal rat submandibular gland salivary protein gene called "common salivary protein 1" (CSP1). Identical clones were also identified in cDNA libraries from adult male parotid, submandibular, and sublingual glands. CSP1 transcripts are at least 10-fold more abundant in the sublingual gland than in the submandibular or parotid glands. In situ hybridization and immunocytochemical localization demonstrated the presence of CSP1 transcripts and proteins in sublingual gland serous demilune cells, parotid and submandibular gland intercalated duct cells, and in the type III (proacinar) cells of the neonatal submandibular gland. This cell-type distribution is similar to that described by Ball and colleagues (Ball, W. D., Hand, A. R., and Johnson, A. O. (1988) Dev. Biol. 125, 265-279) for the developmentally regulated submandibular gland B1-immunoreactive proteins. Immunoblotting of salivary secretion identified proteins of M(r) 20,000 in sublingual, 16,000 in submandibular and 22,000 and 16,000 in parotid gland. The M(r) 20,000 sublingual and 22,000 parotid proteins represent N-glycosylated forms of a M(r) 16,000 apoprotein, suggesting that these salivary proteins arise by post-translational modification of a common precursor.

Molecular cloning of developmentally regulated neonatal rat submandibular gland proteins.

At birth, the rat submandibular gland (SMG) contains two transient secretory cell types that produce several characteristic salivary proteins. Proteins SMG-A, B1, and B2 (23.5, 26 and 27.5 kDa) are products of the neonatal type III cells, but not the adult acinar cells. Protein C (89 kDa), a major product of the neonatal type I cells, is either absent or present at greatly diminished levels in the secretory cells of the adult gland. The decrease in biosynthesis of these neonatal salivary proteins occurs concomitantly with the increase in levels of characteristic adult SMG products. In order to understand these developmentally regulated changes in SMG salivary protein gene expression, we have initiated the molecular cloning and characterization of neonatal submandibular gland proteins from a 5-d-old rat submandibular gland cDNA library. Clones encoding SMG-A were isolated by homology to the mouse parotid secretory protein (PSP). SMG-A was shown to be derived from a salivary protein multigene family that also includes PSP. Cloning and characterization of additional neonatal rat submandibular gland proteins was initiated by screening the 5-d-old rat submandibular gland cDNA library with first strand cDNA prepared from 1-d-old rat submandibular glands. Clones corresponding to a highly abundant 3 kb transcript present in the neonatal rat SMG, but not in adult submandibular, sublingual, or parotid gland have been identified. The size, abundance, and organ specificity of this transcript suggest that it may encode protein C.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of the N region in signal sequence and signal-anchor function.

To determine the role of sequences other than the hydrophobic core in mediating signal sequence function, we examined the behavior of fusion proteins and deletion mutants in cell-free systems. We demonstrate that neither the N nor the C region of the preprolactin signal sequence is necessary for translocation. However, insertion of sequences with either a net charge of +2.5 or -6.0 between the N region and the hydrophobic core of the signal converted it into a signal-anchor. The topologies adopted (types I and II, respectively) were opposite those predicted from the distribution of charges surrounding the hydrophobic core of the signals. When these mutant signals were located in the interior of an otherwise secreted protein, both sequences functioned as stop-transfer sequences. Related mutations were assayed in fusion proteins in which the IgM transmembrane domain functioned as an amino-terminal signal-anchor. For these molecules, the distribution of charged residues surrounding the hydrophobic core had no influence on the topology adopted. Our results suggest that features other than simple charge distribution play an important role in determining membrane topology in vitro.

Neonatal rat submandibular gland protein SMG-A and parotid secretory protein are alternatively regulated members of a salivary protein multigene family.

Submandibular gland-A (SMG-A) is a major secretory product of the neonatal rat submandibular gland but is not synthesized by the acinar cells of the adult gland. The leucine-rich protein is a predominant product of the adult rat parotid gland. Preliminary data had indicated that the leucine-rich protein and SMG-A might be identical proteins whose expression was differently regulated in the parotid and submandibular salivary glands. cDNA clones encoding SMG-A and the leucine-rich protein were identified by homology to a major mouse parotid gland product, parotid secretory protein (PSP), and characterized. The leucine-rich protein shares extensive sequence homology with mouse PSP throughout its 5'-untranslated, protein coding and 3'-untranslated regions, prompting our suggestion that it should henceforth be referred to as rat PSP. SMG-A is more divergent, having greatest identity with rat and mouse PSP in its signal peptide and 3'-untranslated sequences. Transcripts homologous to SMG-A and rat PSP, but more closely related to SMG-A, were identified in rat sublingual gland by Northern blot analysis. These findings indicate that rat SMG-A and PSP arise from alternatively regulated members of a multigene family.

Premature termination of transcription from the P1 promoter of the mouse c-myc gene.

Modulation of transcriptional elongation within the c-myc gene is thought to play a major role in determining levels of c-myc mRNA in both normal and tumor cells. A discrete site of blockage to transcriptional elongation has previously been localized at the 3' end of exon 1 of the mouse and human c-myc genes. We here identify an additional site of transcriptional attenuation that is located between the P1 and P2 promoters of the c-myc gene and that mediates premature termination of transcripts initiating from the P1 promoter. A 95-nucleotide DNA fragment derived from this region prematurely terminated transcription when placed downstream from the promoter of the H-2Kbm1 gene and assayed by expression in Xenopus oocytes. We also show that the previously identified attenuation signal in exon 1 of the mouse c-myc gene can mediate premature termination of P1-initiated transcripts. Premature termination of P1-initiated transcripts presumably increases transcription from the downstream P2 promoter; aberrant regulation of this termination may explain the increased use of the P1 promoter that is characteristic of certain tumors in which myc is overexpressed.

Accumulation and localization of two adult acinar cell secretory proteins during development of the rat submandibular gland.

The seromucous acinar cells of the adult rat submandibular gland secrete a characteristic mucin glycoprotein and a family of unusual glutamine/glutamic acid-rich proteins (GRP). Monoclonal antibodies to the mucin and GRP localized in a very few Type III cells in glands of newborn and 1 day-old rats, using light and electron microscopic immunocytochemistry. Both mucin and GRP reactivities were present in the polymorphic Type IIIP granules during the 1st postnatal week. By 9 days after birth, the granules contained both mucin and GRP and were mucous-like in appearance. At earlier stages, however, cells containing only GRP or mucin could be found, indicating that the initiation of GRP and mucin biosynthesis may not be coordinately regulated. No reactivity was seen in the neonatal Type I cells or in duct cells at any age. Northern and Western blot analysis showed GRP mRNA and protein levels to be barely detectable at birth, with marked increases during the first 2 postnatal weeks. In contrast, Western blots of B1-immunoreactive proteins (B1-IP) showed levels highest in the 1st week and markedly decreased in the adult. Immunocytochemical colocalization, using gold particles of different sizes, showed that the B1-IP, mucin, and GRP colocalized in the granules. These results strengthen the hypothesis that the adult acinar cells develop from the neonatal Type III cells. No evidence was obtained for the involvement of Type I cells in the pathway of acinar cell development.