Linda M. Sabatini

Histatins, a family of salivary histidine-rich proteins, are encoded by at least two loci (HIS1 and HIS2)

We screened a human parotid gland cDNA library with mixed synthetic oligonucleotide probes representing a central coding region common to histatins 1 and 3. Sequence analysis of 12 histatin cDNA clones strongly suggests that the histatin protein family is encoded by at least two closely related loci (HIS1 and HIS2) such that histatins 1 and 3 are primary products of HIS1(1) and HIS2(1) alleles, respectively, and that histatins 4-6 are derived from histatin 3 by proteolysis. We present additional data indicating that histatin 2 may represent the non-phosphorylated form of histatin 1.

Tissue Distribution of RNAs for Cystatins, Histatins, Statherin, and Proline-rich Salivary Proteins in Humans and Macaques

The tissue distribution of the mRNAs for a number of salivary proteins [proline-rich proteins (PRPs), statherin, cystatins, and the histatins] has been examined in humans and macaques in order to investigate their possible functions and tissue-specific regulation. We have shown that PRP RNAs (0.8-1.5 kb) are expressed in human and rhesus parotid and submandibular glands, and in the human bronchus. The genes for the acidic and basic PRPs are differentially regulated in these tissues. RNAs for acidic PRPs are predominantly expressed in the submandibular gland, for basic PRPs in the respiratory tract, and for both acidic and basic PRPs in the parotid gland. Protein studies of secretions from these tissues confirm the RNA results. Statherin RNA (0.65 kb) was detected in human and rhesus parotid and submandibular glands and the human bronchus, as well as in rhesus lacrimal glands. Statherin was found by tissue immunoperoxidase staining in the serous cells of respiratory tract submucosal glands, which is the same location for the synthesis of PRPs. Several cystatin RNAs (0.8-1.3 kb) were differentially expressed in human parotid glands, submandibular glands, and the bronchus, and in lacrimal glands from both rhesus and cynomolgus macaques. RNAs (0.6 kb) for the histatins were found only in parotid and submandibular glands. Thus, it appears that PRPs, statherin, and cystatins may play a broader role in the physiology of biological fluids and secretions than previously suspected, since they are found in secretions other than saliva. However, the functions of the histatins are restricted to saliva. These studies also pose some interesting questions regarding the differential expression of these genes in a variety of secretory tissues.

The human cystatin C gene (CST3) is a member of the cystatin gene family which is localized on chromosome 20

The fourth gene from the human cystatin gene family of salivary-type cysteine-proteinase inhibitors has been isolated and partially characterized by DNA analysis. The gene, which we name CST3, codes for human cystatin C, and has the same organization as the CST1 gene for cystatin SN and the CST2 gene for cystatin SA. Southern analysis of EcoR I digested DNAs from 32 independent somatic cell hybrid clones hybridized to a probe from CST1 demonstrated that all members of the cystatin gene family segregate with human chromosome 20. These results indicate that the genes for salivary-type cystatins and cystatin C are members of a multigene family--the cystatin gene family.

Serial cultivation of epithelial cells from human and macaque salivary glands

SummaryTo study the regulation of human salivary-type gene expression we developed cell culture systems to support the growth and serial cultivation of salivary gland epithelial and fibroblastic cell types. We have established 22 independent salivary gland epithelial cell strains from parotid or submandibular glands of human or macaque origin. Nineteen strains were derived from normal tissues and three from human parotid gland tumors. Both the normal and the tumor-derived salivary gland epithelial cells could be serially cultivated with the aid of a 3T3 fibroblast feeder layer in a mixture of Ham’s F12 and Dulbecco’s modified Eagle’s media supplemented with fetal bovine serum, calcium, cholera toxin, hydrocortisone, insulin, and epidermal growth factor.Salivary gland epithelial cells cultured under these conditions continued to express the genes for at least two acinar-cell-specific markers at early passages. Amylase enzyme activity was detected in conditioned media from cultured rhesus parotid epithelial cells as late as Passage 5. Proline-rich-protein-specific RNAs were detected in primary cultures of both rhesus and human parotid epithelial cells. Neither amylase enzyme activity nor PRP-specific RNAs were detected in fibroblasts isolated from the same tissues. In addition, salivary gland epithelial cells cultured under our conditions retain the capacity to undergo dramatic morphologic changes in response to different substrata.The cultured salivary gland epithelial cells we have established will be important tools for the study of salivary gland differentiation and the tissue-specific regulation of salivary-type gene expression.

Structure and sequence determination of the gene encoding human salivary statherin

Human statherin (STT) is a low-Mr (43 amino acids) acidic phosphoprotein secreted mainly by salivary glands. It acts as an inhibitor of precipitation of Ca.phosphate salts in the oral cavity. DNA (12.2 kb) was isolated from human genomic phage lambda libraries as a series of overlapping clones, and the nucleotide sequence of the STT-encoding gene (STT) was determined. The transcribed region spans 6.5 kb and contains six exons and five introns. Upstream DNA (1.6 kb) was also sequenced and a number of possible regulatory elements were identified. The exon-intron boundaries of the STT gene roughly coincide with the protein-coding regions of the mRNA and with the functional domains of STT. This pattern of organization has been seen in a variety of eukaryotic genes and is consistent with the domain theory of gene evolution.

Survival and tissue distribution of human T-cell clones in SCID mice

Evidence from animal experiments and clinical trials suggests that in vitro expanded T-cell clones could be useful in adoptive therapy of cancer and viral infections. To establish an in vivo model for adoptive therapy with cloned human T cells, we studied the survival and tissue distribution of human αβ CD4 + T-cell clones transplanted intraperitoneally into mice with severe combined immune deficiency (SCID) mice. Four clones, expanded in vitro in recombinant human interleukin-2 (IL-2), were injected into 14 cyclophosphamide-conditioned mice, subsequently inoculated daily with IL-2. Using flow-cytometry analysis, human T cells were detected in the peritoneal cavity wash (PCW) but not in other tissues of 12 mice at 1 to 4 weeks after injection. A reverse transcriptase polymerase chain reaction (RT-PCR) specific for the constant region of human TCR β chain revealed a positive signal in 12 of 14 mice in PCW, eight in spleen, seven in lymph nodes, seven in liver, six in bone marrow, and two in blood. The frequency of human T-cell detection decreased with time. Five to seven sites were positive in mice killed at 1 week, one to four sites at 2 weeks, none to one site at 3 weeks, and three sites at four weeks. Thus human T-cell clones transplanted in SCID mice can survive for at least 4 weeks, even in the absence of specific antigen. The clones migrate at low levels outside the peritoneal cavity ; therefore, the SCID mouse might serve as a model to study adoptive therapy with cloned T cells.

The October 29th Group: Defining a feminist science

Synopsis A group of scientists and those interested in the sciences, all feminists, has been meeting for six years at the University of Wisconsin-Madison to discuss and define a feminist critique of science. We consider science an important area for feminist investigation for several reasons. Scientific research is funded, conducted, and applied for the goals of those with political power. Patriarchy throughout history has used science to justify itself, for example, assigning sociobiological explanations to class, race, or gender inequalities. Many of the characteristics we value would generally be considered part of “dgood” science. These include sharing of information, availability of funding for projects representing diverse points of view, opportunity regardless of gender, race, religion, and judging work on its merits rather than the status of the authors. Other criticisms are more specifically feminist, examining how patriarchal politics and worldview are played out in science. Some examples include an emphasis on the control of nature rather than understanding, a search for “master” molecules presupposing a hierarchical rather than an interactive model, or reliance on reductionism, quantification, and linear thinking while denegating other modes of obtaining and organizing information. We emphasize ways to bring a feminist commitment and philosophy into the laboratory and outline one possible mechanism for transformation of science to more egalitarian principles. Our group represents for us an example of feminist science in action, in its emphasis on open discussion, support as well as criticism, and awareness of the relationship between means and ends, theory and practice.