Sequences of cDNAs for human salivary and pancreatic α-amylases

The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous.Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase.     

Estimation of Type-Specific Neutralizing Antibody to Herpes Simplex Virus Type 2 in Uterine Cervical Cancer Patients by a New Absorption Method

A simple method of estimating type-specific neutralizing antibody to type 2 herpes simplex virus (HSV-2) was devised with the use of the microneutralization system. Serially diluted serum was mixed in the well with a constant amount of type 1 virus (HSV-1), and after 3 days' incubation at 37 C, the plate was irradiated with ultraviolet light. The absorbing HSV-1 consisted of culture fluid plus an extract of infected Vero cells not especially concentrated. The well then received indicator HSV-1 or HSV-2, and after being left at 37 C for 1 hr a suspension of dispersed Vero cells was dropped into the wells, following our standard neutralization procedure. Preliminary tests with rabbit antisera showed that even a low level of HSV-2 antibody was detected by this method, unless an exceptionally high titer of HSV-1 antibody originally coexisted with the HSV-2 antibody. Sera from acutely infected persons testified to the specificity of the antibody so detected. It was revealed by means of the new technique that the rate of HSV-2 antibody was significantly higher in uterine cervical cancer patients than in control women. There was no correlation between the clinical stage of cervical cancer and the presence of HSV-2 antibody.     

Candida albicans and Candida parapsilosis Rapidly Up-Regulate Galectin-3 Secretion by Human Gingival Epithelial Cells

Galectin-3 is a β-galactoside-binding C-type lectin that plays an important role in innate immunity. The purpose of this study was to determine whether Candida albicans and Candida parapsilosis up-regulate galectin-3 secretion by human gingival epithelial cells and gingival fibroblasts. Ca9-22, a human gingival epithelial cell line, and human gingival fibroblasts were incubated in the presence or absence of C. albicans or C. parapsilosis without serum. Levels of secreted human galectin-3 in culture supernatants were measured by enzyme-linked immunosorbent assay. We also pretreated Ca9-22 cells with cytochalasin D (an actin polymerization inhibitor), ALLN (a calpain inhibitor) and LY294002 [a phosphatidylinositol-3 kinase (PI3K) inhibitor] to determine whether the up-regulation of galectin-3 secretion was mediated by cytoskeletal changes, protease activity, or PI3K signaling. Galectin-3 secretion was significantly and rapidly up-regulated by live C. albicans and C. parapsilosis, as well as heat-killed C. albicans. In addition, cytochalasin D, LY294002 and ALLN did not inhibit the up-regulation in galectin-3 secretion. These results suggest that both live and heat-killed C. albicans and C. parapsilosis may increase the activity of the innate immune system and invasion by other microorganisms via up-regulation of galectin-3 secretion.     

Stimulation of anaphylaxis in the mouse footpad by dietary fish oil fatty acids

The effect of fish oil-derived omega-3 (omega-3) fatty acids on anaphylaxis, Arthus and delayed type hypersensitivity reactions in mice has been investigated. Mice on a normal chow diet were fed eicosapentaenoic acid and docosahexaenoic acid at a dose of 500 and 333 mg/kg/day, respectively, by a gastric tube over a period of 61 days. Control groups were given water, safflower oil or oleic acid. Anaphylactic and Arthus type reactions were induced in the mouse footpad using bovine serum albumin as an antigen. Carrageenin was utilized to produce a delayed type hypersensitivity reaction. The animals fed omega-3 fatty acids induced a more anaphylactic foodpad reaction. There was no significant effect of the diet on Arthus and delayed type hypersensitivity responses. There was no effect of the fish oil-supplemented diet on production of antibodies to bovine serum albumin. Synthesis of prostaglandin E2 by peritoneal macrophages was significantly inhibited in the animals fed omega-3 fatty acid-enriched fish oil, while leukotriene B4 production was not affected. These results suggest that a diet enriched in omega-3 fatty acids modulates production of arachidonic acid metabolites and this may influence anaphylaxis, but not Arthus and cellular mediated hypersensitivity responses.     

Effect of forskolin on collagen production in clonal osteoblastic MC3T3-E1 cells

The effect of forskolin on collagen production in osteoblasts was investigated by using clonal osteoblastic MC3T3-E1 cells cultured in a-minimum essential medium containing 0.1% bovine serum albumin. Forskolin increased the adenylate cyclase activity in membranes pelleted from homogenates of the cell line in a dose-dependent manner. The drug caused a 13-fold stimulation at 10(-4) M, indicating that the compound directly acts on adenylate cyclase, leading to an increase in the intracellular cAMP content of the cells. Collagen accumulation in the cultures was elevated by one-day treatment with 5 X 10(-5) M forskolin to about twice that in the controls. The stimulation was mainly due to an elevation in collagen synthesis but not to an inhibition of intracellular collagen degradation because forskolin dose-dependently increased collagen synthesis; it also significantly increased the amount of low-molecular-weight hydroxyproline found in the cultures. Cells treated with forskolin produced mainly type I collagen, as found in bone matrix in situ, with only small amounts of other types of collagen. Furthermore, forskolin time-dependently inhibited DNA synthesis in the cells, indicating that the increase in type I collagen synthesis by forskolin was not due to stimulated cell proliferation. These results suggest that cAMP is closely linked to the differentiation of osteoblasts in vitro.     

Twelve loci form a continuous linkage map for human chromosome 18

We have constructed a primary genetic map of human chromosome 18 consisting of 11 DNA markers and one serological marker (JK). Two of these loci define highly polymorphic VNTR systems. The markers define a continuous genetic linkage map of 97 cM in males and 205 cM in females; female genetic distances in a panel of 59 three-generation families were consistently about twice those observed in males. The high odds in support of the linear order of the markers on this recombination map, and the extent of coverage of chromosome 18, indicate that this map will permit efficient linkage studies of human genetic diseases that may be segregating on chromosome 18 and will provide anchor points for development of high-resolution maps for this chromosome.     

Detection by DGGE of a new polymorphism closely linked to the adenomatous polyposis coli region

Summary The EF5.44 locus is in close proximity to the chromosome 5 region to which the genetic defect responsible for familial adenomatous polyposis has been mapped. We have devised two oligonucleotides that promote the specific polymerase chain reaction (PCR) amplificiation of a 365-bp sequence in this region. Analysis by denaturing gradient gel electrophoresis of the resulting fragment has unravelled individual differences that could be identified as a single base pair change in aMnlI restriction site. This PCR assayable polymorphism increases the informativeness at this locus, and should be useful in the presymptomatic diagnosis of familial adenomatous polyposis.     

A method for measuring the amount of retrogradely transported HRP in the rat masseteric motoneuron using Mesulam's HRP histochemical protocol and an image processing system. An investigation of the effect of dopamine receptor antagonists on the retrograde transport of HRP

We developed a method for a determination of the amount of retrogradely transported HRP in the rat masseteric motoneuron using a modification of Mesulam's HRP histochemical protocol and an image processing system combined with a light microscope and a television camera. To test the validity and reproducibility of the new method, a quantitative comparison of the amount of dark blue granules of HRP-product in the cell body of masseteric motor neurons was performed between the right and left trigeminal motor nuclei of 70 rats, which resulted in no significant difference. An additional study used the method was made of the effects of administration of five dopamine receptor antagonists with different biochemical and pharmacologic properties on retrograde transport of HRP in the rat masseteric motoneuron. As a result, chlorpromazine, haloperidol, SCH 23390, and sulpiride significantly enhanced retrograde transport of HRP as against domperidone which showed no significant change in the transport. A possible regulatory system for retrograde transport of HRP in the masseteric motoneuron was discussed in relation to the action of the dopamine receptor.     

The influence of experimentally produced oligohydramnios on lung growth and pulmonary surfactant content in fetal rabbits.

To study the effect of oligohydramnios on lung growth and biochemical lung development in fetal rabbits, amniotic fluid was drained through a tube inserted into the maternal peritoneal cavity on the 23 day of gestation. Littermate fetuses without an amniotic shunt were used as controls. The fetuses were delivered abdominally on the 28 day of gestation. In a total of 8 pregnant does, 17 fetuses underwent amniotic shunting and 22 fetuses were used as controls. The amniotic shunt produced a significant reduction in the amniotic fluid volume. There were no differences in the wet weights of the fetal body, liver or brain between the two groups. However, the amniotic shunt significantly decreased the wet weight of the fetal lung, fetal lung wet weight/body weight ratio, and protein concentration per lung as compared to the control fetuses. In the fetal liver and brain tissues, no changes were found in the concentrations of total phospholipids, phosphatidylcholine (PC) or disaturated phosphatidylcholine (DSPC, the main component of lung surfactant) per g of wet tissue and per mg of protein. However, the lungs of the fetuses with amniotic shunts contained significantly more PC and DSPC, and the L/S ratio was higher than in the control fetuses. These results suggest that the oligohydramnios produced by an amniotic shunt causes pulmonary hypoplasia, but raises the pulmonary surfactant content of fetal rabbit lung.     

Cytologic diagnosis of laryngeal and hypopharyngeal squamous cell carcinoma in sputum

A prospective study of the value of sputum cytology in the diagnosis of squamous cell carcinoma of the larynx and hypopharynx is reported. Sputum cytology established the diagnosis in 63.5% of the patients with laryngeal lesions and in 77.4% of the patients with hypopharyngeal lesions. In laryngeal cancer, a positive diagnosis by sputum cytology was related to clinical T factors (according to the TNM classification): while only 29.4% of T1 lesions were positively detected by sputum cytology, 63.3% of T2 lesions, 69.7% of T3 lesions and 79.2% of T4 lesions were so detected. In hypopharyngeal cancer, there was no discernible relationship between sputum cytodiagnosis and clinical T factors. Generally, there was only a small number of cancer cells present in the sputum in these cases. Some of the squamous cancer cells were not very conspicuous and would require careful screening of the sputum specimens to be detected.