Salivary proline-rich protein genes on chromosome 8 of mouse.

Endonuclease restriction (Hind III) fragments of DNA from Chinese hamster X mouse somatic cell hybrids hybridized with proline-rich protein complementary DNA clones only when the DNA was isolated from cells containing mouse chromosome 8, or a fragment of chromosome 8. The evidence suggests that proline-rich protein genes are located at the proximal portion of chromosome 8 toward the centromere.

Measurement of circulating desialylated glycoproteins and correlation with hepatocellular damage.

Addition of increasing amounts of (125)I-labeled desialylated thyroxine-binding globulin (DTBG) to hepatic cell membranes resulted in a progressive increase in binding. Saturability of membrane sites was indicated by a concentration beyond which further increases in [(125)I]DTBG resulted in no further binding. The binding curve for [(125)I]DTBG was similar to binding curves of desialylated orosomucoid, fetuin, and ceruloplasmin. An inhibition assay system using hepatic cell membranes showed that desialylated orosomucoid had a greater affinity for membrane binding sites than did DTBG but desialylated fetuin and ceruloplasmin bound less avidly than DTBG. Serum from normal persons and patients with a variety of illnesses was tested for its ability to inhibit [(125)I]DTBG binding. The inhibitory activity of 1 ml of normal serum was equivalent to that of 0.2-2 mug DTBG. Patients with Laënnec's cirrhosis, biliary cirrhosis, and hepatic metastases had greatly increased inhibitory activity in their serum. Patients with jaundice due to extrahepatic obstruction had inhibitory activity not significantly different from that found in normal serum. Column chromatography of normal serum on Sephadex G-200 resulted in inhibitory activity throughout the range of protein molecular weight. Desialylation of normal serum with neuraminidase enhanced the inhibitory activity but did not change the distribution of the activity. Gel chromatography of cirrhotic serum showed markedly increased inhibitory activity associated with the macroglobulins and the 4.5S peak and a new peak of inhibitory activity in the low molecular weight area was also seen. Inhibition of desialylated glycoprotein binding to liver cell membranes by serum from patients with hepatocellular disease raises the possibility that desialylated serum glycoproteins accumulate in the circulation and that patients with compromised hepatocellular function may no longer be able to clear them from the circulation. Alternatively, accumulation of desialylated glycoproteins in the circulation could result from defective protein synthesis by the diseased liver.

Gene expression following direct injection of DNA into liver.

The liver is an attractive target tissue for gene therapy. Current approaches for hepatic gene delivery include retroviral and adenoviral vectors, liposome/DNA, and peptide/DNA complexes. This study describes a technique for direct injection of DNA into liver that led to significant gene expression. Gene expression was characterized in both rats and cats following injection of plasmid DNA encoding several different proteins. Luciferase activity was measured after injection of plasmid DNA encoding the luciferase gene (pCMVL), beta-galactosidase (beta-Gal) activity was evaluated in situ using plasmid DNA encoding Lac Z (pCMV beta), and serum concentration of secreted human alpha-1-antitrypsin was measured following injection of plasmid DNA encoding this protein (pRC/CMV-sHAT). Several variables, including injection technique, DNA dose, and DNA diluent, were investigated. Direct injection of pCMVL resulted in maximal luciferase expression at 24-48 hr. beta-Gal staining demonstrated that the majority of transfected hepatocytes were located near the injection site. Significant concentrations of human alpha-1-antitrypsin were detected in the serum of animals injected with pRC/CMV-sHAT. These findings demonstrate the general principle that direct injection of plasmid DNA into liver can lead to significant gene expression.

Proline-rich proteins and glycoproteins: expression of salivary gland multigene families.

Our recent research interests have focused on a group of unusual proteins and glycoproteins high in proline content, or the so-called proline-rich proteins (PRPs). The PRPs are tissue-specific expressions of salivary gland multigene families. Normally PRPs are not detected or are present in very low amounts in rat, mouse and hamster salivary glands, but these unusual proteins are dramatically induced by treatment with the catecholamine isoproterenol. The structures and organizations of several PRP mRNAs and PRP genes have been determined. The amino acid sequences of all PRPs show 4 distinct regions, namely, a signal peptide, a transition region, a repeat region and a carboxyl-terminal region. Glycoproteins induced by isoproterenol treatment may be N-glycosylated or O-glycosylated. The N-glycosylated glycoprotein GP-158 from rat submandibular glands has a 12 amino acid glycopeptide which repeats possibly 49 times. Proline-rich proteins of the parotid glands of rats and mice are also greatly induced by dietary tannins. The apparent unique occurrence of PRPs in saliva suggests that one biological role is to neutralize the detrimental effects of dietary tannins and other polyphenols. The upstream regions of the mouse and hamster PRP genes contain cyclic AMP-regulated sequences as demonstrated by deletions and transient transfections. The PRP multigene family members of mouse are all located on chromosome 8.

Regulation of proline-rich protein gene expression by cyclic AMP in primary cultures of hamster parotid glands.

Proline-rich proteins (PRPs) constitute a group of unusual salivary proteins encoded by tissue-specific multigene families which can be dramatically induced (20- to 70-fold) in vivo in rats, mice and hamsters by treatment with the beta-agonist isoproterenol. Addition of dibutyryl cyclic AMP (dbcAMP) or forskolin to hamster parotid gland primary cultures resulted in a large increase (15- to 30-fold) in PRP mRNA levels. The same time-course and levels of induction of PRP mRNA by dbcAMP and isoproterenol were found in primary cultures, indicating that both effectors act through the same mechanism. Induction by isoproterenol, but not by dbcAMP or forskolin, was blocked by the beta-antagonist propranolol. Incorporation of [3H]proline into PRPs was stimulated in primary cultures by all three effectors. The greatest increase in proline incorporation was in the [3H]PRPs recovered in the culture medium of induced cells. These studies demonstrate that cAMP or agents which increase intracellular cAMP levels increase PRP gene expression in primary cultures of parotid glands. Pretreatment of the cells with cycloheximide blocked the induction of PRP mRNAs which indicates that the synthesis of a trans-acting factor may be necessary for transcriptional activation of the PRP genes. alpha-Amylase mRNA, another tissue-specific gene product, was not significantly affected by cycloheximide treatment.

Expression, regulation, and function of the SPR family of proteins. A review.

The small, proline-rich (SPR) genes consist of three subclasses closely linked on human chromosome 1, a region referred to as the epidermal differentiation complex. SPR genes consist of two exons, with the second exon containing the entire open reading frame. SPRs are expressed in all squamous tissues of the skin, scalp, footpad, vaginal epithelia, and most of the epithelial lining of the digestive tract, including the lip, tongue, esophagus, and forestomach. Although SPR1 is absent in normal mucociliary epithelium of the respiratory tract, epithelia that undergo squamous differentiation in response to vitamin-A deficiency or to injury owing to exposure to environmental toxicants express SPR1. High levels of SPR1 are detected in various diseases and cancers of the skin or respiratory epithelia and in nonkeratinizing papillary adenocarcinomas. SPR expression can be regulated by transcriptional factors, by posttranscriptional factors, or by factors that affect SPR1 mRNA translation or protein turnover. Furthermore, regulation can be affected by the state of cell proliferation. The presence of SPR1 in most of these epithelia, and the absence of SPR3 in normal skin, suggest that these subclasses have distinct functions. Various approaches to the study of the cross-linked envelope (CE) components in identifying SPR1 and SPR2 and in suggesting that SPRs are one of the precursor proteins of the CE. However, expression of SPR1 in nonsquamous tissues and cell lines indicates a function not associated with squamous differentiation. Several studies have demonstrated that SPR1 antibodies react with nuclear proteins and that SPR1 is expressed in cells before entering the G0 phase of the cell cycle. Future studies should clarify the role of SPRs by modifying their contents in CE, and should identify SPR-associated proteins to clarify the cell growth-related role of SPR1.

Functional status of patients with end-stage renal disease.

A longitudinal study of 979 patients with end-stage renal disease from 27 dialysis centers in the Upper Midwest was conducted to measure the patients' functional status with use of the Karnofsky Activity Scale. At the initiation of dialysis, 50% of all patients were rehabilitated or caring for themselves, and the three variables that most influenced the initial rehabilitation score were age, diabetic status, and sex. Initial functional status was also analyzed for three cohorts of dialysis patients, grouped according to outcome (renal transplantation, continued dialysis, and death). Patients who received a renal transplant had initial rehabilitation scores that were higher than those who underwent dialysis for 2 years or those who died. In the group of patients who underwent dialysis for 2 years, a statistically significant improvement in rehabilitation scores was noted at 2 years in comparison with the scores obtained at the initiation of dialysis. Initial rehabilitation scores were good predictors of the 2-year scores. Of the patients in the 2-year dialysis cohort, 78% maintained or had improvement in their functional status.

Hospitalization in dialysis patients.

During 1981, 946 patients with advanced renal failure who were maintained by dialysis were studied to assess the frequency and the duration of hospitalizations and to identify complications that prompted hospitalization. Five hundred fifty-eight patients (59%) were hospitalized for a total of 1,207 times (mean of 1.8 stays/yr and 15 days/yr at risk for the entire group). The major reasons for hospitalization were dialysis access problems (25%), gastrointestinal complications (13%), and cardiac complications (13%). Both the rate of stays/yr and the rate of days/yr increased with advancing age and were highest in patients who died during the year of study. Both the rate and the duration of hospitalization were higher for patients maintained by peritoneal dialysis than for those on hemodialysis (P less than 0.001). In patients younger than 45 years of age, diabetics had more frequent and more prolonged hospitalizations than nondiabetics, whereas in those 46 to 60 years of age, complications other than diabetes predisposed to hospitalization. In those 61 years of age or older, in whom hospitalization rates were the highest, no single risk factor could be identified as predisposing to hospitalization other than age and peritoneal dialysis. Although the interactions of these factors were not assessed, considerable agreement existed with previous studies that had been analyzed in a more sophisticated manner.

Acoustically induced cortical arousal increases phasic pharyngeal muscle and diaphragmatic EMG in NREM sleep.

Six healthy subjects (3 males, 3 females) were studied to assess phasic inspiratory responses of upper airway (UA) and diaphragm muscles to electrocortical arousal independent of other potential respiratory stimulation. Transient electroencephalographic (EEG) arousal (abrupt EEG frequency shift > or = 3 s without awakening) was induced during supine stage 2 non-rapid-eye-movement (NREM) sleep with binaural tone bursts (0.5 s, 4 kHz, 25-95 dB). Electromyograms (EMG) of levator veli palatini (EMGlvp) and genioglossus (EMGgg) were obtained with intramuscular electrodes, and EMG of diaphragm (EMGdi) was obtained with esophageal electrodes. EMG signals were processed as moving time-averaged inspiratory activity over 100-ms windows. For each arousal, each of five consecutive postarousal breaths (R1-R5) was scored for peak inspiratory phasic EMG and normalized as percent averaged EMG of the three prearousal breaths for all muscles. After arousal, EMGlvp was increased for R1-R5 and EMGgg and EMGdi were increased for R1-R4. The increase in EMGlvp was greater than those of EMGgg and EMGdi for all response breaths. There was a significant increase in EMGlvp in all subjects, and EMGgg and EMGdi were significantly increased in three and two subjects, respectively. These data indicate that isolated transient electrocortical arousal is generally associated with phasic inspiratory recruitment of UA and diaphragm muscles in normal humans during NREM sleep; velopharyngeal muscle recruitment appears to be more consistent and of greater magnitude and duration than that of oropharyngeal muscle or diaphragm. We speculate that transient arousal from sleep may contribute to UA patency independent of chemical and mechanical respiratory stimuli.

A corpus-linguistic investigation of dental English.

This article begins by presenting corpus linguistics and explaining how the corpus approach to language analysis can be used to investigate the language of dentistry. It then reports findings from an analysis that was carried out with readily available software and a corpus of 1,400 dental research abstracts. Included is information about word frequency in dental abstracts and also about how certain words tend to co-occur in sentences and form regular patterns. The article concludes with a discussion of ways that corpus-based findings can be applied to the teaching of English to non-native speakers of the language, many of whom will need English skills for their future careers in dentistry.

Salivary proline-rich proteins: biochemistry, molecular biology, and regulation of expression.

The proline-rich proteins (PRPs) in mammalian salivary glands are encoded by tissue-specific multigene families whose members have diverged with respect to structure and regulation of expression. PRPs are expressed constitutively in humans, and comprise about [70%] of the total salivary proteins. Families of similar proteins are dramatically increased or induced in parotid and submandibular glands of rats, mice and hamsters by treatment with the [beta-] agonist isoproterenol. Feeding tannins to rats and mice mimics the effects of isoproterenol on the parotid glands. Salivary PRPs may constitute a defense mechanism against tannins and other polyhydroxylated phenols ingested. Putative transcriptional regulatory sequences have been identified in mouse PRP genes.

Glycoproteins: carbohydrates to cloning.

This article is dedicated to Professor Saul Roseman and briefly outlines some of the early studies on sialyltransferases, on glycoproteins such as pig submaxillary mucins and, more recently, on a series of unusual proteins and glycoproteins high in proline, the so-called proline-rich proteins. Hopefully, it represents, in an inadequate manner, my appreciation for 'The man and his works'. During the Roseman Symposium at the 11th International Symposium on Glycoconjugates in Toronto, several of his former students and postdocs tried to describe what it was like in the Roseman laboratory. Clearly, the time I was in Saul's lab was like no other time in my career. Thanks for everything, Saul.

Characterization of vitellogenin from white sturgeon, Acipenser transmontanus.

Sturgeon are an ancient family (Acipenseridae) of fishes that lie close to the divergence of fish that eventually evolved into terrestrial animals and those that evolved into modern teleost species. Therefore, white sturgeon vitellogenin sequences fill a gap in the current understanding of the functional domains of this protein family. Vitellogenin cDNA was sequenced and used to investigate gene expression in white sturgeon, Acipenser transmontanus. Estrogen-induced vitellogenin mRNA was detected in the livers of both males and females and was also detected in undifferentiated gonads of estrogen-treated fish. Low levels of vitellogenin mRNA were also detected in the testis of both control and estrogen-treated males. The cDNA encoded a 186-kDa protein that was missing only six to seven of the amino-terminal amino acids. Comparisons to silver lamprey, Xenopus, and chicken vitellogenin sequences indicate that the overall structure of the yolk protein domains were highly conserved. There was considerable homology in three regions of the lipovitellin I domain. These conserved sequences are likely to be involved in vitellogenin receptor binding. The phosvitin domain of white sturgeon vitellogenin contained fewer and shorter serine repeats as predicted from yolk protein phosphate content of fish compared to Xenopus and chicken. However, the vitellogenin of white sturgeon had a lower serine content as compared with silver lamprey, indicating that an increased serine content is not strictly a function of evolutionary age.

Regulation of proline-rich protein and alpha-amylase genes in parotid-hepatoma hybrid cells.

Salivary proline-rich proteins are encoded by tissue-specific multigene families, and are dramatically induced in mice, rats, and hamsters by treatment with the beta agonist isoproterenol. Salivary gland cells, however, are difficult to maintain in a differentiated state in culture and can be induced to synthesize proline-rich protein mRNAs for only a few days. In an attempt to establish a cell line in which it would be possible to regulate proline-rich protein gene transcription, rat parotid gland cells were fused with the rat hepatoma cell line, FTO-2B. Fused cells were obtained that had a frequency of 7.5 x 10(-6), which is about 125-fold greater than the reversion rate of FTO-2B. The hybrid cells exhibited induced proline-rich protein mRNA synthesis when incubated with either dibutyryl cyclic AMP or forskolin. The ability to induce these genes was maintained for at least 20 passages. Most of the fused cell populations also synthesized elevated levels of alpha-amylase mRNA, another tissue-specific gene.

Cell cycle-specific expression of G(0)SPR1 in Chinese hamster ovary cells.

A family of small proline-rich proteins (SPR1s) is induced in cells undergoing squamous differentiation. Because SPR1 mRNA is detected in mesenchymal nasal cells of rats exposed to cigarette smoke, expression of this mRNA in other nonsquamous cells and tissues was investigated. Using PCR, low levels of SPR1 mRNA were identified in a number of nondifferentiating cell lines and in nonsquamous tissues. G(0)SPR1 mRNA, the hamster homologue of SPR1 mRNA, was increased 10-fold in Chinese hamster ovary (CHO) cells when the culture reached 80-90% confluence and was downregulated after cells ceased growing at 100% confluence. The deduced amino acid sequence of G(0)SPR1 showed a high homology to the family of SPR1 from different species. Affinity-purified antibodies to SPR1 reacted to about 50% of the CHO cell population, indicating that the protein is expressed at specific stages of the cell cycle. CHO cells that were switched to low-serum medium when they were at 60% confluence showed an increase in G(0)SPR1 levels before the cells entered G0, indicating that G(0)SPR1 may b a signal to cells entering G0. Because expression of the SPR1 family of proteins is associated with squamous differentiation, the observations in the nondifferentiating CHO cells indicate that these proteins may play a role in mediating the withdrawal from the cell cycle prior to the commitment to differentiation.