Deep lamellar keratoplasty by deep parenchyma detachment from the corneal limbs.

AIM: To improve the deep lamellar keratoplasty technique. METHOD: For the easy and reliable perfomance of deep lamellar keratoplasty (DLKP), detachment of Descemet's membrane through the corneal limber flap was improved. To expose Descemet's membrane, the parenchyma was detached by hydrodelamination through a sclerocorneal flap made in the corneal limbs. The parenchyma was removed after the pseudochamber between it and Descemet's membrane was maintained with viscoelastic material. The corneal graft was placed with a running suture. 22 eyes were treated. RESULTS: Complete exposure of Descemet's membrane was obtained in 20 of the 22 eyes (91%). The membrane was perforated in five of the 22 eyes (23%) during surgery, and two of the 22 eyes (9%) were converted to penetrating keratoplasty. These two eyes developed keratoconus after acute corneal hydrops. CONCLUSION: Compared with the conventional procedure, this new method provides easy, reliable exposure of Descemet's membrane.

Unique inhibitory action of the synthetic compound 2-[N-(2-aminoethyl)-N-(5-isoquinolinesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (CKA-1306) against calcium/calmodulin-dependent protein kinase I.

A newly synthesized compound, 2-[N-(2-aminoethyl)-N-(5-isoquinolinesulfonyl)]amino-N-(4-chlorocinnamyl )-N-methylbenzylamine (CKA-1306), was found to inhibit cyclic AMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase I (CaMK I) with IC50 values of 1.6+/-0.14 and 2.5+/-0.16 microM, respectively. In contrast, the established PKA inhibitors H-8 and H-89 inhibited CaMK I with relatively high IC50 values of >100 and 24.4+/-3.2 microM, respectively. An additional inhibitor, KN-62, against Ca2+/calmodulin-dependent protein kinase II (CaMK II) did not inhibit either PKA or CaMK I at the concentrations tested. In our library of many isoquinolinesulfonamide derivatives, only CKA-1306 inhibited CaMK I to a satisfactory degree, suggesting a unique mode of action. Indeed, the inhibition of CaMK I by CKA-1306 was competitive in every respect to Mg2+/ATP, peptide substrate (syntide-2), and Ca2+/calmodulin. This phenomenon may be understood from the context of the recently determined structure of the enzyme in its autoinhibited state. Such kinetic analysis was also extended to cases using a phosphorylated and activated enzyme at Thr177 or a constitutively active, COOH-terminal truncated mutant at Gln293. CKA-1306 still competed with Mg2+/ATP for the two enzymes, but it no longer achieved any competitive advantage over syntide-2. These results may reflect some differences in the active conformation of CaMK I. However, the compound should be constant in its recognition of an Mg2+/ATP-binding site of the enzyme. Though CKA-1306 is not specific to CaMK I, the compound will be useful in studying the enzyme further under limited conditions.

Effect of oestradiol replacement in ovariectomized chickens on pituitary LH concentrations and concentrations of mRNAs encoding LH beta and alpha subunits.

Plasma concentrations of oestradiol and LH and steady-state pituitary concentrations of the mRNAs encoding the LH beta and alpha subunits in the gland were measured at intervals of 1 month for 5 months in female chickens. In addition, feedback regulation by oestradiol of concentrations of mRNAs encoding these LH subunits was studied in ovariectomized young (3-week-old) and adult hens. Concentrations of the mRNAs encoding the LH beta and alpha subunits increased significantly before sexual maturity and the changes were highly correlated with increases in plasma and pituitary concentrations of LH. Ovariectomy significantly reduced circulating plasma concentrations of oestradiol but increased pituitary concentrations of mRNAs encoding LH beta and alpha subunits and pituitary and plasma concentrations of LH in adult laying hens. In addition, in 3-week-old chickens, ovariectomy induced an increase in plasma and pituitary gland concentrations of LH. This ovariectomy-induced mRNA expression and tissue and plasma increases of LH were prevented by oestradiol replacement. These data suggest that a negative feedback mechanism by ovarian oestradiol inhibits LH release from the pituitary and pituitary LH biosynthesis in chickens.

Isolation and comparison of rat cDNAs encoding Ca2+/calmodulin-dependent protein kinase I isoforms.

For possible multiple isoforms of Ca2+/calmodulin-dependent protein kinase I (CaM kinase I), only one cDNA (CaM kinase I alpha) hitherto has to been cloned. By screening of embryonic (E18) rat brain cDNA libraries, we have now isolated two additional examples (CaM kinase I beta and gamma). Northern blot analysis revealed CaM kinase I alpha to predominate over I beta and gamma in rat brain. Analysis of the tissue distribution of the isoforms by reverse transcription-polymerase chain reaction (RT-PCR) protocols demonstrated CaM kinase I alpha in a variety of tissues while the expression of CaM kinase I beta and gamma was more limited to the brain. The obtained results support the idea that CaM kinase I exists as a set of isoforms.

HMN-709, a chlorobenzenesulfonamide derivative, is a new membrane-permeable calmodulin antagonist.

Our objective is to describe the basic chemical and biological properties of the new calmodulin antagonist HMN-709 (2-[N-(2-aminoethyl)-N-(4-chlorobenzenesulfonyl)]amino-N-(4-flu orocinnamyl)-N-methylbenzylamine). This newly synthesized compound was found to inhibit the Ca2+/calmodulin-dependent activation of calmodulin kinase I, smooth muscle myosin light chain kinase and Ca2+-phosphodiesterase with IC50 values of 1.57+/-0.21, 2.29+/-0.09 and 0.30+/-0.08 microM (mean+/-S.E.), respectively. This compound showed little or no effect on the Ca2+/calmodulin-independent activation of protein kinase A, protein kinase C and basal phosphodiesterase. In addition, HMN-709 inhibited calmodulin kinase I competitively with respect to calmodulin (Ki=0.88 microM) and non-competitively with respect to ATP. Affinity chromatography, with HMN-709-coupled Sepharose HP, showed that the compound bound to calmodulin in a Ca(2+)-dependent manner and did not bind to calmodulin kinase I. These results suggest that HMN-709 antagonizes calmodulin by binding to Ca2+/calmodulin. HMN-709 inhibited collagen-induced platelet aggregation with an IC50 value of 11.80+/-0.86 microM (mean+/-S.E.) without inhibiting phorbol 12,13-dibutyrate-induced aggregation at doses up to 12 microM. HMN-709 appears to be a new, membrane-permeable calmodulin antagonist that may be used for studying the involvement of calmodulin in cellular processes.

Prolactin, growth hormone, and luteinizing hormone-beta subunit gene expression in the cephalic and caudal lobes of the anterior pituitary gland during embryogenesis and different reproductive stages in the chicken.

Messenger RNA (mRNA) levels of prolactin (PRL), growth hormone (GH), and luteinizing hormone (LH)-beta subunit in the cephalic and caudal lobes of the anterior pituitary gland of the chicken during embryonic development and different reproductive stages were determined by Northern blot analysis and are related to hormone levels in plasma and each lobe. Steady-state levels of 1.4-kb transcript of PRL of the cephalic and caudal lobes increased on Days 18 and 20 of incubation, respectively. The levels were greater in the cephalic lobe than in the caudal lobe on Days 18 and 20 of incubation. Levels of PRL in each lobe show similar changes to those of mRNA levels. GH mRNA levels of both lobes started to increase on Day 16 of incubation but the levels were higher in the caudal lobe than in the cephalic lobe on Days 16-20 of incubation. Levels of GH in the cephalic lobe increased discontinuously, while those in the caudal lobe increased continuously on days of incubation. LH-beta mRNA was not detected in embryos. Levels of PRL mRNA increased approximately 18-, 20-, and 3-fold in hens at 1 and 3 weeks of egg incubation and brooding stages in the hen, relative to prelaying, respectively. These levels were higher in the cephalic lobe than in the caudal lobe at all stages except for prelaying stages. Levels of PRL in each lobe displayed essentially similar changes to the mRNA levels. GH mRNA levels did not significantly vary during the cycle but were higher in the caudal lobe than in the cephalic lobe. There was no difference in LH-beta mRNA levels between the two lobes but the levels were higher at prelaying and laying stages than at incubation and brooding stages.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycerol oxidase, a novel copper hemoprotein from Aspergillus japonicus. Molecular and catalytic properties of the enzyme and its application to the analysis of serum triglycerides.

Glycerol oxidase purified from Aspergillus japonicus AT 008 had Mr = 400,000 and contained 1 mol of protoheme IX and 2 g atoms of copper/mol of enzyme protein. The absorption maxima of the oxidized form were found at 557, 530, 420, 280, and 238 nm, and those of the reduced form at 557 and 430 nm. Anaerobic addition of glycerol to the enzyme produced both a shift of the Soret band from 420 to 410 nm and bleaching of the alpha and beta bands at 557 and 530 nm. The ESR spectrum of glycerol oxidase showed three major signals at g = 1.99, g = 2.00, and g = 2.02. The signals at g = 1.99 and g = 2.02 were diminished by the anaerobic addition of glycerol, and the three signals completely disappeared after the addition of either dithionite or diethyldithiocarbamate. Exposure of glycerol oxidase to a borate buffer of pH 10.0 resulted in activation of the enzyme with concomitant enhancement of the ESR signals at g = 1.99 and g = 2.02. Since glycerol oxidase acts predominantly on glycerol, the enzyme can be employed in a specific colorimetric assay for serum triglycerides in combination with lipoprotein lipase.

Purification and properties of lipase from Rhizopus japonicus.

Lipase [EC 3.1.1.3] was purified from the culture supernatant of Rhizopus japonicus KY 521 by ethanol precipitation, chromatography on Column-lite, affinity chromatography on heparin-Sepharose 4B, and separation into two lipases, I and II, by isoelectric focusing. The purified lipases I and II were found to be homogeneous by disc electrophoresis, and showed isoelectric points at pH 7.4 and pH 7.9, respectively. They both had an apparent molecular weight of about 42,000, hydrolyzed tricaprin very rapidly, and exhibited a pH optimum around pH 7.0-8.5. These lipases were inhibited by the addition of serum to the reaction mixtures. These lipases were enhanced slightly in the absence of serum by high concentrations of NaCl and protamine, but were inhibited strongly by these compounds in the presence of serum.

Oxidation of 3beta-hydroxyandrostenes by the 3beta-hydroxy-steroid oxidase (cholesterol oxidase) from Brevibacterium sterolicum prior to their analysis by gas-liquid chromatography-mass spectrometry.

The 3beta-hydroxysteroid oxidase from Brevibacterium sterolicum has been applied to the oxidation of a number of 3beta-hydroxyandrostenes, including polar steroids containing up to three other hydroxylic groups. The substrates, products, and derivatives thereof have been examined by gas-liquid chromatography. Retention index increments for these conversions, and for parallel transformations of other steroids, show considerable regularities, and together with mass spectrometric data afford characteristic structural information.