Preliminary study of etidronate for prevention of corticosteroid-induced osteoporosis caused by oral glucocorticoid therapy.

Glucocorticoids (GCs) are widely used for the treatment of various diseases, particularly in dermatology. However, there have been few reports about the outcome of treatment for GC-induced osteoporosis in patients with dermatological conditions receiving oral GCs. The present study was undertaken to prospectively evaluate the usefulness of etidronate for preventing steroid-induced osteoporosis in patients on prolonged GC therapy as routine clinical management. In total, 110 patients receiving oral GC therapy were enrolled into the study. Of these, 87 patients were evaluated (44 patients with collagen diseases, 13 patients with autoimmune bullous dermatoses, 19 patients with chronic eczema/dermatitis, 2 patients with toxicoderma/drug eruption and 9 others). Urinary deoxypyridinoline (DPD) was evaluated as a marker of bone resorption, and serum bone-specific alkaline phosphatase (BAP) as a marker of bone formation. Significant increases in urinary DPD were seen in the control group after oral GC therapy had been continued for ≥ 1 year. Treatment with etidronate suppressed this increase. When the patients were stratified according to gender, this improvement was more obvious in women. No significant difference in serum BAP level was found between the two groups. These results suggest that bisphosphonates may be useful for preventing steroid-induced osteoporosis in dermatology patients (particularly women) receiving oral GC therapy.

Expression of alpha-amylase in human lung cancers.

Thirty-three human lung tumors were studied for the expression of alpha-amylase by immunohistochemical and Northern blot analyses. Twenty of them were adenocarcinomas, among which 17 proved to be adequate for mRNA analyses and were, except for two, amylase mRNA producers. Seven were squamous cell carcinomas, none of which produced amylase. The remaining six consisted of two undifferentiated small cell carcinomas, and one each of undifferentiated large cell carcinoma, carcinoid tumor, mucoepidermoid carcinoma, and metastatic lung cancer; the mucoepidermoid carcinoma proved to be an amylase producer. These observations strongly suggest that among lung cancers, the production of alpha-amylase is a property commonly associated with adenocarcinomas and can be used for distinguishing cell types. Histogenesis and carcinogenesis in lung cells are discussed in connection with the cells that produce amylase.

Hydrolysis of low-density lipoprotein phospholipids in arterial smooth muscle cells.

The hydrolysis of phosphatidylcholine (PC) associated with low-density lipoprotein (LDL) by homogenates of smooth muscle cells from rabbit aorta was studied. 1-Palmitoyl-2-[14C]oleoylPC associated with LDL (LDL-P[14C]OPC) or 1-linoleoyl-2-[14C]linoleoylPC associated with LDL (LDL-L[14C]LPC) was used as the substrate. The optimum pH for the formation of [14C]oleoyllysoPC from LDL-P[14C]OPC and for the formation of [14C]linoleoyllysoPC from LDL-L[14C]LPC was pH 4.5, and pH 4.5 and 7.0, respectively. These activities were designated as phospholipase A1 activities. The optimum pH values for the formation of [14C]oleate from LDL-L[14C]OPC and for the formation of [14C]linoleate from LDL-L[14C]LPC were pH 4.5 and 6.5, and pH 4.5, 6.5 and 8.5, respectively. These activities were designated as phospholipase A2 activities. Ca2+ did not affect acid phospholipase A1 activity, but decreased acid phospholipase A2 activity for the hydrolysis of LDL-L[14C]LPC. When smooth muscle cells were incubated with LDL, both phospholipase A1 and phospholipase A2 activities at pH 4.5 for the hydrolysis of LDL-L[14C]LPC increased significantly. These results indicate that phospholipases A1 and A2, which hydrolyze PC associated with LDL, exist in arterial smooth muscle cells and are involved in the metabolism of LDL incorporated into these cells.

Corrected sequences of cDNAs for human salivary and pancreatic alpha-amylases [corrected].

The nucleotide sequences of the cloned human salivary and pancreatic alpha-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 homology 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 alpha-amylase cDNAs with those previously obtained for mouse alpha-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human alpha-amylase.

Cloning, characterization and nucleotide sequences of two cDNAs encoding human pancreatic trypsinogens.

Two cDNA clones encoding two major human trypsinogen isozymes were isolated from a human pancreatic cDNA library. The deduced amino acid (aa) sequences of the two trypsinogen precursors are found to have 89% sequence homology, and have the same number of aa (247), including 15 aa for a signal peptide and 8 aa for an activation peptide. Southern blot analysis of human genomic DNA using the cloned cDNA as a probe, revealed that the human trypsinogen genes constitute a multigene family of more than ten genes.

Expression of human salivary alpha-amylase gene in Saccharomyces cerevisiae and its secretion using the mammalian signal sequence.

A cDNA fragment coding for human salivary alpha-amylase precursor was joined to the promoter of the Saccharomyces cerevisiae PHO5 gene, and the recombinant gene was inserted into a vector plasmid capable of autonomous replication in yeast. Yeast cells transformed with this recombinant plasmid synthesized about 5 X 10(5) molecules of the enzyme per cell when synthesis was induced by deprivation of inorganic phosphate and released about half of the synthesized enzyme into the medium. The enzyme is stable, and exhibited the same specific activity as alpha-amylase in human saliva. The amylase-producing yeast grew on starch and produced alcohol.

Primary structure of human pancreatic alpha-amylase gene: its comparison with human salivary alpha-amylase gene.

We have determined the entire structure of the human pancreatic alpha-amylase (Amy2) gene. It is approx. 9 kb long and is separated into ten exons. This gene (amy2) has a structure very similar to that of human salivary alpha-amylase (Amy1) gene [Nishide et al. Gene 41 (1986a) 299-304] in the nucleotide sequence and the size and location of the exons. The major difference lies in the fact that amy1 has one extra exon on the 5' side. Other differences are at the 5' border of exon 1 and the 3' border of exon 10. The close similarity of these two genes, as compared with mouse pancreatic and salivary amylase genes, suggests that during evolution, the divergence into the two amylase genes may have occurred after the divergence of mice and man.

Overlapping two genes in human DNA: a salivary amylase gene overlaps with a gamma-actin pseudogene that carries an integrated human endogenous retroviral DNA.

The human salivary amylase gene (amy1), consisting of eleven exons, is expressed in the salivary gland and in some amylase-producing tumors. Its uppermost exon and the following intron, along with the 5'-flanking region of this gene, are shown to be superimposed with a gamma-actin pseudogene sequence, a portion of which is transcribed into salivary amylase mRNA and another portion of which serves as a promoter for the amy1 gene. In the further upstream region, the gamma-actin pseudogene sequence is interrupted by a human endogenous retroviral nucleotide sequence.

Primary structure of human salivary alpha-amylase gene.

A recombinant clone which covers the human salivary alpha-amylase gene in a single insert has been isolated from a human genomic DNA library using a human salivary alpha-amylase cDNA as a probe. Restriction mapping and nucleotide (nt) sequence analysis revealed that this gene is approx. 10 kb long and is separated into eleven exons by ten introns. Its 5'-flanking region has some sequence homology with that of mouse salivary alpha-amylase gene [Schibler et al., J. Mol. Biol. 155 (1982) 247-266].

Production of salivary type alpha-amylase in human lung cancer.

alpha-Amylase, which is produced by lung cancer tissue, was studied by cloning cDNAs from a cell line originating from lung cancer that produces amylase. Sequencing studies with this cDNA showed that the expressing gene is of the salivary type. The specific location of the start point of transcription, as revealed by S1 mapping, supported this conclusion.

Effects of chloroquine on the metabolism of phosphatidylcholine associated with low density lipoprotein in arterial smooth muscle cells.

Phospholipid associated with LDL (LDL-phospholipid) has been suggested to affect metabolism of LDL in arterial smooth muscle cells. However, the metabolism of LDL-phosphatidylcholine in these cells has not been well clarified. We compared the metabolic pathway of LDL-phosphatidylcholine with that of cholesteryl ester associated with LDL (LDL-cholesteryl ester) in rabbit arterial smooth muscle cells by incubating the cells in the absence or presence of chloroquine, an inhibitor of lysosomal function. When the cells were incubated with LDL-[3H]cholesterol linoleate in the absence of chloroquine, 26.6 and 51.4% of incorporated radioactivity was found as cholesteryl ester in the lysosome-rich and microsome-rich fractions, respectively. When the cells were incubated in the presence of 50 microM chloroquine, the radioactivity found as cholesteryl ester in the lysosome-rich fraction increased to 45.5% while that in microsome-rich fraction decreased to 21.4%, indicating that LDL-cholesteryl ester accumulated in lysosomes as a consequence of inhibition of lysosomal function. When the cells were incubated with LDL-[14C]linoleoyl phosphatidylcholine in the absence of chloroquine, 25.1% of incorporated radioactivity was found as phosphatidylcholine in the lysosome-rich fraction and 24.8% in the cytosol-rich fraction. When the cells were incubated in the presence of chloroquine, phosphatidylcholine-associated radioactivity found in the lysosome-rich and cytosol-rich fractions changed only to 28.8% and 26.1%, respectively, showing that LDL-phosphatidylcholine did not accumulate in lysosomes when lysosomal function was inhibited. In conclusion, these data indicate that LDL-phosphatidylcholine, in contrast to LDL-cholesteryl ester, is not only hydrolyzed in lysosomes, but also at other subcellular sites.

A familial hyperalphalipoproteinemia with low uptake of high density lipoproteins into peripheral lymphocytes.

A patient with an extremely high level of high density lipoprotein (HDL)-cholesterol and HDLc-like particles in the serum is discussed. The patient was a 46-year-old female with a serum total cholesterol concentration of 382 mg/dl and HDL-cholesterol level of 214 mg/dl. The HDL-cholesterol levels of her mother, brother, sister and 2 of her daughters were 82 mg/dl, 82 mg/dl, 74 mg/dl, 82 mg/dl and 82 mg/dl, respectively (mean HDL-cholesterol levels of control subjects: 52 +/- 6 mg/dl in males and 55 +/- 8 mg/dl in females). Her serum apolipoprotein A-I and E levels were elevated. Zonal ultracentrifugal analysis of her serum lipoproteins showed that the increased level of HDL-cholesterol was mainly due to HDL2; HDLc-like particles were also recognized between the LDL and HDL fractions. The incorporation of the patient's HDL and HDLc-like particles into cultured HepG2 cells was almost the same as that of HDL (1.063 less than d less than 1.21) from normal control serum. The incorporation of normal control HDL into the patient's peripheral blood lymphocytes was markedly less than that into lymphocytes from normal controls. These findings are discussed in terms of the reason for hyperalphalipoproteinemia in this patient.

Regression of coronary atherosclerosis by combined LDL-apheresis and lipid-lowering drug therapy in patients with familial hypercholesterolemia: a multicenter study. The LARS Investigators.

The purpose of the LDL-Apheresis Regression Study (LARS) group, which included 13 institutions in Japan, was to investigate the effects on coronary atherosclerosis of LDL-apheresis combined with cholesterol-lowering drugs. Changes in coronary artery stenosis were assessed angiographically in 37 patients with familial hypercholesterolemia (7 homozygotes and 25 heterozygotes) and hypercholesterolemia which had not been defined as familial hypercholesterolemia (5 patients) by visual judgement and computer analysis. Definite regression was observed in 14 cases, including 4 homozygotes and 10 heterozygotes and others. Regression occurred as often in patients with severe coronary artery disease (2 or more vessel disease) as in those having less severe disease. Our results encourage initiation of aggressive cholesterol-lowering therapy to produce regression of coronary atherosclerosis in FH patients at high risk for cardiovascular events.

Effects of intensive lipid lowering by low-density lipoprotein apheresis on regression of coronary atherosclerosis in patients with familial hypercholesterolemia: Japan Low-density Lipoprotein Apheresis Coronary Atherosclerosis Prospective Study (L-CAPS).

Twenty-five heterozygous familial hypercholesterolemic patients treated with LDL-apheresis and drugs and 11 patients treated with drugs underwent follow-up angiography 2.3 years later. One-hundred thirteen lesions were measured by quantitative angiography. Mean LDL-cholesterol levels during the trial were 140 +/- 34 mg/dl in the apheresis group and 170 +/- 58 mg/dl (P < 0.05) in the control group. The mean changes in minimal lumen diameter of lesions were +0.19 +/- 0.30 mm (improved) in the apheresis group (n = 76) and -0.44 +/- 0.40 mm (worsened) in the control group (n = 37) (P < 0.0001). When progression and regression were defined as a change in minimal lumen diameter of +/- 0.67 mm, in the apheresis group, two (8%) patients had progression, 19 (76%) stayed unchanged and four (16%) had regression, but in the control group seven (64%) patients had progression and four (36%) stayed unchanged. The frequency of regression or no change was significantly higher in the apheresis group than in the control group (P < 0.004). Intensive cholesterol lowering therapy with LDL-apheresis and lipid lowering drugs can achieve a substantial decrease in LDL-cholesterol levels to induce regression of coronary lesions in familial hypercholesterolemic patients with advanced coronary artery disease.

Abdominal wall fat index, estimated by ultrasonography, for assessment of the ratio of visceral fat to subcutaneous fat in the abdomen.

PURPOSE: To establish a new index of regional fat distribution using ultrasonography for assessment of the ratio of the visceral fat area (V) to the subcutaneous fat area (S) (V/S ratio). SUBJECTS AND METHODS: The subjects examined were 62 patients (23 males and 39 females); 51 patients had hyperlipidemia and 11 patients had glucose intolerance. The mean body mass indices ranged from 20.3 to 42.9. The mean age of the patients was 44 +/- 13 years. The thicknesses of the preperitoneal fat layer (P) and subcutaneous fat layer (S) in the abdomen were measured by ultrasonography and the P/S ratio was calculated. The V/S ratio was obtained with radiographic computed tomography. RESULTS: Of the various P/S ratios examined, the ratio of the maximum thickness of preperitoneal fat to the minimum thickness of subcutaneous fat was most closely correlated with the V/S ratio (r = 0.746, p < 0.0001). This ratio was termed the abdominal wall fat index (AFI). AFI was positively correlated with serum triglyceride levels and negatively correlated with high-density lipoprotein cholesterol (r = -0.312, p < 0.05), whereas the V/S ratio was correlated with triglyceride levels. AFI was positively correlated with basal insulin levels in both men and women. CONCLUSION: These results suggest that AFI measured by ultrasonography may be a new indicator of visceral fat deposition, and may reflect metabolic disorders such as lipid metabolism and glucose metabolism disorders.

Effect of simvastatin on the lipid profile of hemodialysis patients.

BACKGROUND: Simvastatin, a 3-hydroxy 3-methylglutaryl co-enzyme A (HMG-CoA) reductase inhibitor, is used widely for treatment of hypercholesterolemia. Simvastatin may be a suitable treatment for dyslipidemia in hemodialysis (HD) patients. However, investigation of the side-effects and safety of long-term administration of simvastatin to HD patients has been limited. In this study, we investigated the effects and safety of simvastatin and its effects on lipoprotein metabolism in hypercholesterolemic patients on HD. METHODS: Simvastatin was administered at a dosage of 5 mg/day for 24 weeks to 38 HD patients with high serum total cholesterol (TC) levels (200 mg/dl) or low high-density lipoprotein cholesterol (HDL-C) levels (35 mg/dl). Every four weeks, serum lipids, apolipoprotein, lipoprotein (a) [Lp(a)] and malondialdehyde (MDA) levels were measured. In addition, lipid levels were determined in each lipoprotein fraction separated by ultracentrifugation. RESULTS: After 24 weeks of simvastatin administration, TC significantly decreased by 25.7%, and low-density lipoprotein cholesterol (LDL-C) was significantly decreased by 33.6%. Triglyceride (TG) and HDL-C showed no significant changes. Apolipoprotein (apo) B significantly decreased by 24.5% and apo E by 30.0%. No significant changes were observed in the other apolipoproteins. MDA was also significantly decreased, whereas Lp(a) was not significantly altered. In the lipoprotein fractions, very LDL cholesterol (VLDL-C), intermediate-density lipoprotein cholesterol (IDL-C), LDL1 cholesterol (LDL1-C), and LDL2 cholesterol (LDL2-C) showed significant decreases. No particular side-effects were observed during the 12 months of simvastatin administration. CONCLUSIONS: These results suggest that simvastatin appears to be safe and effective in HD patients with hypercholesterolemia.

Effects of pindolol on serum lipoproteins and postheparin lipolytic activities in hypertensive patients.

Thirty-four hyperlipoproteinemic, hypertensive patients received 5 mg of pindolol twice daily for 12 weeks. During pindolol administration, there were significant decreases in serum triglyceride levels and increases in high-density lipoprotein cholesterol (HDL-C) levels, while total cholesterol levels did not change. Serum levels of very-low-density lipoprotein (VLDL) triglyceride and VLDL cholesterol decreased over time as HDL-C increased. There was a significant increase in low-density lipoprotein cholesterol at week 12. Apolipoprotein (apo) A-I, A-II, and B levels did not change during pindolol administration, but apo C-II, C-III, and E levels decreased significantly. Lipoprotein lipase activity in heparin-treated plasma was significantly higher after pindolol administration. The results suggest that the reduction in triglyceride levels and increase in HDL-C after pindolol are partly a response to an increase in the hydrolysis of VLDL resulting from an increase in lipoprotein lipase activity.

Serum lipid levels in hypertensive patients during captopril treatment.

Captopril (37.5 mg daily) was administered to 64 hypertensive patients for 16 weeks. During treatment, systolic and diastolic blood pressures decreased significantly (from means of 164/98 mmHg before treatment to 150/90 mmHg at four weeks and 142/86 mmHg at eight weeks; P less than 0.001), but serum levels of total cholesterol, triglycerides, lipoprotein cholesterol, lipoprotein triglyceride, and apolipoproteins showed no significant changes. Scores on the atherogenic index did not change. Patients with high initial total cholesterol levels and low high-density lipoprotein cholesterol levels tended to improve their lipid levels. It is concluded that captopril does not adversely affect serum lipoprotein metabolism.

High prevalence of herpes simplex virus type 2 in acute retinal necrosis syndrome associated with herpes simplex virus in Japan.

PURPOSE: To determine the type of herpes simplex virus in acute retinal necrosis syndrome associated with herpes simplex virus. METHODS: Herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, Epstein-Barr virus, and cytomegalovirus were examined by polymerase chain reaction in intraocular specimens from 16 patients with acute retinal necrosis syndrome. Anti-herpes simplex virus type 1 and anti-herpes simplex virus type 2 type-specific antibodies in serum from the patients were detected by enzyme immunoassay. RESULTS: Of 16 patients with acute retinal necrosis syndrome, seven were polymerase chain reaction positive for herpes simplex virus type 2 and nine were positive for varicella-zoster virus. Anti-herpes simplex virus type 2 antibody was positive and anti-herpes simplex virus type 1 antibody was negative in the sera of the seven patients with herpes simplex virus type 2 DNA-positive acute retinal necrosis syndrome. In contrast, anti-herpes simplex virus type 2 antibody was absent in all nine varicella-zoster virus DNA-positive acute retinal necrosis syndrome patients. CONCLUSION: Herpes simplex virus type 2 has been demonstrated to be the major causative agent in acute retinal necrosis syndrome associated with herpes simplex virus by molecular biological and serological methods. Negative preexisting anti-herpes simplex virus type 1 antibody may play an important role in acute retinal necrosis syndrome associated with herpes simplex virus type 2.

Effect of intracameral anesthesia on the corneal endothelium.

PURPOSE: To evaluate the effects of intracameral anesthesia on the corneal endothelium. SETTING: Department of Ophthalmology, Yokohama City University School of Medicine, Yokohama, Japan. METHODS: This study comprised 24 eyes of 12 white rabbits. One eye of 3 rabbits each was injected with preservative-free lidocaine at concentrations of 0.02, 0.2, or 2% and the fellow eye injected with balanced salt solution (BSS) as a control. The anesthetic agent was injected into the anterior chamber using a bimanual technique. Immediately after enucleation, the cornea was examined by scanning electron microscopy. RESULTS: Scanning electron microscopy revealed no abnormal findings in the eyes injected with lidocaine 0.02 or 0.2% when compared with eyes in the control group. Scanning electron microscopy of the eyes injected with lidocaine 2% showed irregular hexagonal endothelial cells and a significant loss of microvilli. CONCLUSION: Intracameral anesthesia with high concentrations of lidocaine risks corneal endothelial damage but at the low concentration usually used in cataract surgery did not appear to have an adverse effect.