alpha-Amylase expressed in human liver is encoded by the AMY-2B gene identified in tumorous tissues.

BACKGROUND: An alpha-Amylase in human liver is detected with an anti-human salivary amylase antibody, but the enzyme activity is very low. We previously found that the rat liver contained an amylase which differed from the enzyme of mice. In this study, we characterized the human liver amylases biochemically and immunohistochermically. METHODS AND RESULTS: Although the amylase activity of human liver was much lower than that of rat, protein moiety and sugar chains of the human amylase were identified as similar to the rat liver enzyme with an anti-human salivary amylase antibody and by concanavalin A (Con A) affinity chromatography. Liver amylases from human and rat were the same size, 50 kDa, on Western blot analysis and had the same isoelectric points. The cytoplasm of hepatocytes was moderately stained immunohistochemically with the anti-human salivary amylase antibody. Intrahepatic bile ducts were also stained weak-to-moderately. RT-PCR, with a specific primer for the consensus sequence of human amylases, amplified a single 474-bp product from the human liver total RNA. The PCR product was sequenced and referred to the homology. Thirteen bases in the 434-bp fragment of the human liver amylase differed from the corresponding region of the AMY-1 gene transcript and the deduced amino acid sequence differed at five residues. The human liver amylase cDNA sequence was identical to the corresponding cDNA of the AMY-2B, which was known to expressed in tumorous tissues. In situ hybridization revealed the expression of AMY-2B mRNA in non-tumorous human liver. CONCLUSIONS: The present results suggest the possibility that a novel amylase detected in tumorous tissues and encoded by the AMY-2B gene is a liver-specific amylase expressed in the human liver.

Expression of alpha-amylase gene in rat liver: liver-specific amylase has a high affinity to glycogen.

The reactivity of rat liver alpha-amylases with maltotriose (G3), maltopentose (G5) and glycogen has been investigated. Liver amylases were found to be glycosylated and to have a molecular mass of 50 kDa by Western blotting using an anti-human salivary amylase antibody. The glycosylated liver amylases were found to be capable of G3- and G5-hydrolysis and of glucose formation, as demonstrated by thin-layer chromatography. When the amylase preparation was exposed to different concentrations of glycogen and run on a cellulose acetate membrane, the mobilities of rat liver amylases significantly decreased with tailing directly from the point of origin. In contrast, rat salivary amylases were not so much. These results indicate that rat liver amylases have a strong affinity to glycogen. We confirmed the expression of liver-specific amylases in rat liver by reverse transcriptional-polymerase chain reaction (RT-PCR); PCR products showed one band of an expected size of 474 bp using primers tested in the present study. A partial nucleotide sequence was then determined. When compared with the gene of mouse liver amylase, the substitution of 26 bases out of 434 bases was elucidated. The present data demonstrate the presence of liver-specific amylases in rats.

Glycosylated salivary alpha-amylases are capable of maltotriose hydrolysis and glucose formation.

The physiological and/or clinical significance of sugar chains in human salivary alpha-amylase was investigated in terms of substrate-specificity for synthesized malto-oligosaccharides. Glycosylated and non-glycosylated alpha-amylases were prepared on a Sephacryl S-200 column, in which the amylases were separated into four fractions from the different affinities for Sephacryl: fraction I, amylases bearing sugar chains with sialic acid; fraction II, amylases bearing sugar chains without sialic acid; fractions III and IV, non-glycosylated amylases. These were classified according to the differences in their affinities for lectins, molecular sizes and isoelectric points. The inhibitory effect of maltotriose (G3) on starch hydrolysis of the amylase fraction, suggests that starch and G3 can be the substrate for glycosylated amylase, and that the glycosylated amylases are capable of G3 hydrolysis for conversion into maltose and glucose. Using malto-oligosaccharides, G3, G4, G5 and G7, as substrates, the substrate-specificities and G3/G5 ratio of amylase activities in the four fractions were examined. Maltopentaose, G5, is routinely used as a substrate for alpha-amylase, and then we assumed that both glycosylated and non-glycosylated amylases react with G5. Moreover, the results indicate that the glycosylated amylases clearly had a higher capacity for G3 hydrolysis than the non-glycosylated amylases, although no substrate preference of either type of amylase was observed among G4, G5 and G7. Glycosylated amylases have the capacity for glucose formation from malto-oligosaccharides.

Total synthesis of (+/-)-acetomycin and design of esterase-resistant analogs.

The synthesis of acetomycin and related analogs was investigated. Acetomycin was synthesized from diethyl allyl(methyl)malonate in 6.5% yield over 18 steps. The total number of steps was improved compared to our previous synthesis; i.e., four steps shorter, and the total yield was 4.5% greater than the previous synthesis. Acetomycin analogs with benzoyloxy and pivaloyloxy groups, instead of an acetoxy group at the 5-position of the gamma-butyrolactone ring were designed as esterase-resistant models and prepared similarly. Although they showed a similar level of cytotoxicity as acetomycin in vitro, they were not resistant to porcine liver esterase, and lost cytotoxicity in vivo.

Somatic mosaicism for a DMD gene deletion.

Mosaicism is a mixed state, with two cell populations of different genetic origins caused by a cell mutation occurring after fertilization. In the present case, DNA analysis of lymphocytes led to a DMD diagnosis before death. Postmortem immunocytochemical and DNA analysis showed somatic mosaicism. At age 18 years, blood lymphocyte DNA analysis showed a DMD gene deletion, upstream from exon 7 to the 5' end containing both muscle and brain promoters. As the patient's mother and elder sister had no deletions, he was considered to have a new mutation. Immunocytochemical studies of postmortem tissues showed that dystrophin was absent from the tongue, deltoid, intercostal, psoas and rectus femoris muscles, but there was a mix of dystrophin-positive and negative fibers in the rectus abdominis, cardiac, temporalis and sternocleidomastoid muscles. All diaphragm cells were dystrophin positive. Polymerase chain reaction (PCR) amplification from all tissues except the temporalis and sternocleidomastoid muscles, diaphragm and kidney, in which no deletion was found, showed the deletion from at least exon 6 to the 5' end containing both muscle and brain promoters. In this case, a genomic deletion of the DMD gene contributed to the formation of tissues derived from both ectoderm and endoderm, and cells of mesodermal origin showed genotypic and phenotypic heterogeneity. Our results indicate a mutation of the present case may have occurred just before the period of germ layer formation.

[Molecular genetics of Duchenne/Becker muscular dystrophy].

The X-linked gene responsible for Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) encodes dystrophin, a high-molecular-weight cytoskeletal protein. The identification of the dystrophin gene through positional cloning, and the subsequent description of its protein product have opened several new fields of research and genetic diagnosis. Studies in our laboratory revealed that 26 out of 47 (55%) cases of DMD and nine out of 12 (75%) cases of BMD exhibited genomic deletion. The DMD phenotype is associated with mutations that shift the reading frame of the message, whereas the BMD phenotype is associated with mutations that maintain the reading frame. Immunofluorescence microscopy has established dystrophin's distribution on the plasma membrane of muscles. DMD patients demonstrate a lack of dystrophin on their muscle cell membrane, whereas BMD patients produce a limited amount of protein or abnormally sized protein. Extensive studies on dystrophin and the gene may lead to an understanding of the cause for this and may allow development of a rational treatment for DMD to be developed.

Megacystis and megacolon in an infant with Ehlers-Danlos syndrome.

We report a 14 month old male infant with Ehlers-Danlos syndrome who became 'anuric' due to an acutely dilated urinary bladder. Although the patient was also found to have megacolon, no diverticulum was seen in his gastrointestinal tract or urinary bladder. In order to decompress the urinary bladder and colonic wall, we put an indwelling urinary catheter in place for 2 months, and carried out daily glycerin enema for 3 months. All urological and gastrointestinal symptoms subsided with this intensive medical treatment. The diagnosis of megacystis and megacolon was made very early in life for this patient. This may indicate that the striking extension of gastrointestinal and bladder wall may lead to the development of diverticula of gastrointestinal and urinary tracts in later life.

[Clinical significance of dystrophin test for patients with various neuromuscular diseases--immunofluorescence and immunoblot analyses of dystrophin abnormalities].

The dystrophin test was performed on skeletal muscle specimens from 81 cases with various neuromuscular diseases by using two new monoclonal antibodies. The results were compared with those obtained by using four polyclonal antibodies. These monoclonal and polyclonal antibodies were raised against various portions of the dystrophin molecule. On immunohistochemical analysis, the two new monoclonal antibodies showed the same staining pattern as the four polyclonal antibodies. Non-specific immunostaining of the cytoplasm, often seen with polyclonal antibodies, was not observed with monoclonal antibodies. With the application of monoclonal antibodies, the connective tissue sometimes showed non-specific immunostaining which originated from the second fluorescent antibody. On immunoblot analysis, one of the two monoclonal antibodies, antibody 4-4 C 5, showed weak immunoreactivity, and the 400 kDa dystrophin band was not detected. Three cases out of 15 with Duchenne muscular dystrophy (DMD), and one case out of 3 with limb-girdle type muscular dystrophy which had previously been diagnosed on the basis of clinical data, were found to have non-dystrophin-related muscular dystrophy, and Becker muscular dystrophy (BMD), respectively. Three and two of five cases were diagnosed as DMD and BMD, respectively, though clinical diagnosis had not been possible because they were too young. Clinical diagnosis of congenital muscular dystrophy was confirmed in 9 patients by the dystrophin test. Only one of three certain DMD carriers had a so-called mosaic staining pattern. We conclude that all six antibodies are useful tools for the diagnosis of neuromuscular diseases, because of their high specificity for dystrophin.

["Dismembered spiral flap pyeloplasty" for uretero-pelvic junction obstruction].

We report a new operative technique for plastic correction of uretero-pelvic junction (UPJ) obstruction: dismembered spiral flap pyeloplasty. It is similar to the method described by Culp & DeWeerd in that a flap is made spirally but different in that UPJ is detached. Relatively a long and wide spiral (oblique) flap, the apex of which directs cranioventrally or craniodorsally, is made using the dilated pelvis. The apex of the flap is reflected downward and anastomosed to the split ureteral end. Because the flap is made obliquely, one side of the flap base is approximated to the opposite pelvic margin; this helps to make gradual funnelling of pelvio-ureteric transition. The method seems to be fit for cases with considerably long stenosis of UPJ and with the UPJ locating relatively close to the medial margin of the renal parenchyma. Seven of 26 pyeloplasties were done by this method in our institute, and all the 7 cases had satisfactory results.

Voiding dysfunction in patients with human T-lymphotropic-virus-type-1-associated myelopathy.

Voiding dysfunction in patients with human T-lymphotropic-virus-type-1-associated myelopathy (HAM) was studied. All the patients were diagnosed as having HAM by neurologists. We have already reported on 16 consequent patients with HAM. Almost all of these patients had frequency, and many had urge incontinence of urine and difficulty on voiding. Urodynamic study revealed that their voiding symptoms seemed to be due to detrusor hyperactivity and detrusor-sphincter dyssynergia. However, we have recently treated 2 patients who had a different bladder function. They had both frequency and difficulty in voiding but without urgency. In the urodynamic study both patients did not have involuntary bladder contraction during the filling phase and could not void voluntarily. The reason why these 2 patients had an underactive detrusor is unclear. The fact that the average duration of HAM in the 16 patients previously mentioned was longer than that of the latter 2 patients may suggest that overactivity of the bladder is not prominent in the early phase of this disease.

Effects of inositol 1,4,5-trisphosphate on calcium release from the endoplasmic reticulum and Golgi apparatus in mouse mammary epithelial cells: a comparison during pregnancy and lactation.

It has been established that inositol 1,4,5-trisphosphate(IP3) is responsible for the mobilization of calcium(Ca2+) from intracellular locations in a wide variety of tissues, and that this response triggers the stimulation of several hormones and neurotransmitters. However, these phenomena have yet to be examined in the mammary epithelium. Ca2+ uptake from the medium into the endoplasmic reticulum(ER) and Golgi apparatus in vitro in both pregnant and lactating mouse mammary epithelial cells was studied and a strong Ca2+ release from these organelles into the medium with the use of IP3 was shown. The Ca2+ uptake and its release due to IP3 was also usually greater during pregnancy than lactation.

The activation of phosphatidylinositol-specific phospholipase C by insulin in mammary epithelial cells of lactating mouse.

We have previously demonstrated in vitro that, in the endoplasmic reticulum and Golgi apparatus of mammary epithelial cells of lactating and pregnant mice, inositol 1,4,5-trisphosphate releases Ca2+ that has been stored in these organelles. In this study, we examined whether insulin and prolactin, essential for the growth of mammary gland and for lactation, influenced the activity of phosphatidylinositol-specific phospholipase C in mammary cells. In the plasma membrane fraction of mammary epithelial cells of the DDY mouse strain 5 days after the start of lactation after the first pregnancy, and with phosphatidylinositol as substrate, it was shown that the activity of phospholipase C was enhanced by about four times in the presence of insulin compared with the control. Such enhancement was not found in the membrane fraction treated with prolactin.

[Changes in lipase activity during pregnancy and lactation in the mouse mammary gland].

Mammary gland lipase activity of the mouse increased 45-fold compared to that in unmated gland at the 15th day of pregnancy and was 65-fold at the 20th day of pregnancy. After parturition, the activity abruptly decreased during 3 days to 38% of that at the 20th day of pregnancy. On the other hand, only a very small lipoprotein lipase activity was observed in the pregnant gland, the activity increased to 15-fold that of 20 day pregnancy at the 3rd day of lactation. These facts suggest that the mammary epithelial cells (mammary gland lipase activity was detected only in epithelial cells) utilize the fat reserved in the gland during pregnancy, but the lactating mammary epithelial cells utilize the fat supplied from the blood circulation. Mammary gland lipase activity was decreased by treatment with epinephrine which increased the fat mobilization in other adipose tissues. Hydrocortisone and prolactin decreased the mammary gland lipase activity in the glands of pregnancy and lactation. In addition, no hormone-sensitive lipase activity was observed in the mammary gland. Thus, the control of fat mobilization in the mammary gland must be different from that in other adipose tissues.

Voiding dysfunction in patients with human T-lymphotropic virus type-1-associated myelopathy (HAM).

Voiding dysfunction was evaluated in 16 of 17 patients with human T-lymphotropic virus type-1 associated myelopathy (HAM). Among 16 patients 11 had both frequency and difficulty in voiding and 12 had urge incontinence of urine. Sensation of vesical filling and voiding remained either intact or only slightly suppressed in all patients. Residual urine ranging from 50 to 320 ml was observed in 14 patients. Urodynamic studies showed in every patient an overactive bladder with involuntary detrusor contraction. Only 2 patients had detrusor sphincter synergia, 6 patients had complete detrusor sphincter dyssynergia and 8 patients had incomplete detrusor sphincter dyssynergia. An overactive bladder associated with detrusor sphincter dyssynergia and less affected vesical sensation seems to be a characteristic urodynamic finding in HAM. These results combined with other neurological findings suggest that in patients with HAM lateral columns of the spinal cord between sacral and ponsmesencephalic micturition centre are mainly affected and that the posterior columns of the spinal cord are relatively less affected.

Clinical picture of HTLV-I associated myelopathy.

Clinical and electrophysiological findings in 16 consecutive cases of HTLV-I associated myelopathy are reported. Symmetric spastic paraparesis associated with only mild hyperreflexia in the arms, clinical and electrophysiological evidence of moderate posterior column involvement at the thoracic level, urinary frequency and urgency associated with the detrusor-urethral sphincter dyssynergia but without systemic autonomic involvement, and absence of the segmental grey matter involvement, all suggest the presence of diffuse lesions of the white matter predominantly involving the thoracic cord. We conclude that HTLV-I associated myelopathy is a chronic diffuse leukomyelitis predominantly involving the thoracic cord.

[High resolution ultrasound examination in the diagnosis of the undescended testis in the inguinal region].

Twenty-six undescended testes in 22 patients between 1 and 31 years old were evaluated with ultrasonographic examination between October, 1981 and December, 1985. All 22 patients were operated on, and the accuracy of the ultrasonic diagnosis was evaluated in comparison to that of palpation diagnosis. Fourteen of 26 testes could be palpated preoperatively and these were all identified ultrasonographically and surgically. Twelve testes could not be palpated. On these 12 testes, surgical exploration revealed 9 testes in the inguinal region and absence of 3 testes. Ultrasound examination predicted its presence in 5 of 9 testes and its absence in all 3 testes. Both sonography and palpation failed to identify their presence in 4 tests. Thus, sensitivity of ultrasound examination was 82.6%, specificity 100% and accuracy 84.6% retrospectively. We conclude that ultrasound examination is useful in diagnosis of impalpable undescended testes in inguinal region.