Human peptidylarginine deiminase type III: molecular cloning and nucleotide sequence of the cDNA, properties of the recombinant enzyme, and immunohistochemical localization in human skin.

Peptidylarginine deiminase catalyzes the post-translational modification of proteins through the conversion of arginine to citrulline in the presence of calcium ions. In rodents, peptidylarginine deiminase has been classified into four isoforms, types I, II, III, and IV, which are distinct in their molecular weights, substrate specificities, and tissue localization. Of these isoforms, only type III was detected in epidermis and hair follicles. Although the role of this enzyme in these tissues is not yet clear, indirect data have shown that several structural proteins such as filaggrin, trichohyalin, and keratin are substrates for peptidylarginine deiminase. In this study, we cloned the full-length cDNA of human peptidylarginine deiminase type III (3142 bp) from cultured human keratinocytes by reverse transcription-polymerase chain reaction and by rapid amplification of cDNA ends methods. This cDNA contained a 1995 bp open reading frame encoding 664 amino acids (Mr = 74 770). To explore the physicochemical and enzymatic properties of human peptidylarginine deiminase type III, we constructed a plasmid for producing a recombinant human peptidylarginine deiminase type III in bacteria. The enzymatic characteristics of the recombinant enzyme were very similar to those of the rodent peptidylarginine deiminase type III. The recombinant enzyme showed the catalytic activities toward structural proteins of epidermis and hair follicle, filaggrin and trichohyalin, in which the deiminations maxima of about 60% and 13% arginine residues were observed in filaggrin and trichohyalin, respectively. An immunohistochemical study of human scalp skin with a monospecific anti-peptidyl-arginine deiminase type III antibody revealed that the type III enzyme was localized to the inner root sheath and outer root sheath of hair follicles. Peptidylarginine deiminase type III in the inner root sheath was notable between supramatrix and keratogenous zone and was scarcely detected in cornified hair zone. The enzyme was also expressed in the cuticle layer of hair. On the other hand, expression of the enzyme in the epidermis was very low. These data imply that human peptidylarginine deiminase type III is the predominant isoform in hair follicles and may function as a modulator of hair structural proteins, including trichohyalin during hair and hair follicle formation.

Blood pressure control during eye surgery under local anesthesia using pulse oximetry.

We measured the arterial blood pressure and percutaneous O2 saturation (SpO2) in 39 elderly patients (age range, 70-89 years) who underwent cataract surgery under local anesthesia. Nineteen patients showed a 20% or greater increase in the systolic arterial pressure (SAP) measured every minute in the operating room compared to the basal value measured in the ward. We treated elevation of the SAP by intravenous bolus injection of 0.25 mg nitroglycerin (NTG), followed by continuous infusion at 0.1-0.3 microg/kg/min. NTG caused a statistically significant but clinically irrelevant decrease in SpO2 in the patients breathing room air. Two of these patients showed extremely decreased SpO2. Our results suggest that NTG does not typically cause a severe decrease in oxygenation. It might be advisable, however, for patients with severe lung dysfunction or ischemic heart disease to receive prophylactic O2 administration.

Trichohyalin expression in skin tumors: retrieval of trichohyalin antigenicity in tissues by microwave irradiation.

BACKGROUND: The antitrichohyalin antibody AE 15 is effective for identifying the cell lineage that undergoes the pathway of inner root sheath-type differentiation. Unfortunately, the AE 15 does not react with trichohyalin in tissue that is formalin-fixed and embedded in paraffin according to routine procedures. METHODS: We attempted to retrieve the trichohyalin antigenicity in formalin-fixed, paraffin-embedded biopsy specimens that included normal skin as well as skin tumors such as trichofolliculoma and pilotricoma. RESULTS: We found that the use of a metal solution in combination with microwave oven heating improves the trichohyalin immunoreactivity substantially. Further, trichohyalin was found to be expressed not only in the secondary hair structure in trichofolliculoma but also in a certain cell lineage that differentiates to squamoid cells in pilomatricoma. CONCLUSIONS: Our findings established that surgical specimens processed under routine procedures can be successfully investigated with AE 15 using the microwave irradiation method. Studies of epidermal diseases expressing trichohyalin should provide valuable insights into our understanding the functional significance of trichohyalin during abnormal keratinization.

Microinjection of alpha-calcitonin gene-related peptide into the hypothalamus activates sympathetic outflow in rats.

Effect of rat alpha-calcitonin gene-related peptide (alpha-CGRP) microinjected into various hypothalamic nuclei on plasma levels of catecholamines and arterial blood pressure were investigated in urethane-anesthetized rats. alpha-CGRP (0.05 and 0.25 nmol) microinjected into the hypothalamic paraventricular nucleus (PVN) increased the plasma level of noradrenaline (NA), but not that of adrenaline (AD), in a dose-dependent manner. A similar increase in plasma level of NA was also observed by alpha-CGRP (0.05 nmol) microinjected into the preoptic area (POA), anterior hypothalamus (AH), dorsomedial hypothalamus (DMH) and ventromedial hypothalamus (VMH). A significant increase in arterial blood pressure was observed by microinjection of alpha-CGRP (0.05 nmol) into the PVN, POA, AH and DMH, and the most prominent increase was caused by its microinjection into the PVN. Microinjection of the same dose of this peptide into the VMH, lateral hypothalamic area and posterior hypothalamus was without effect. The increase in plasma level of NA induced by alpha-CGRP (0.05 nmol) into the PVN was not affected by bilateral adrenalectomy. Electrical stimulation of the PVN elicited increases in plasma levels of both NA and AD. This increase in NA was abolished by chemical sympathectomy with 6-hydroxydopamine (50 mg/kg, i.v., 3 days before experiments). These results suggest that activation of the PVN by electrical stimulation elicits both sympathetic and adrenomedullary outflow. alpha-CGRP microinjected into the PVN selectively activates the sympathetic outflow.

Blood pressure control during eye surgery under local anesthesia. Actual status of blood pressure and experience with a new control method using intravenous nitroglycerin.

Blood pressure during eye surgery under local anesthesia was measured in 186 cases treated in our department. 58 cases showed an increase in blood pressure before the operation in spite of premedication; in 32 of these cases there was a 40% rise in blood pressure. Moreover, 38 cases showed at least one ECG abnormality just before the operation in spite of no past history of heart disorders. To control the blood pressure in these cases, intravenous injection of nitroglycerin (Millisrol) was performed continuously and immediately stabilized the blood pressure.

Wavelength dependence of the geometric and structural photoisomerization of bilirubin bound to human serum albumin.

The two quantitatively important photoisomers in bilirubin metabolism during phototherapy are (ZE)-bilirubin and (EZ)-cyclobilirubin. We describe in vitro studies on the wavelength dependence for the geometric (delta 4Z, delta 15Z----delta 4Z, delta 15E) and structural (endovinyl cyclization) photoisomerization of bilirubin bound to human serum albumin by a high performance liquid chromatography method. For the geometric photoisomerization from (ZZ)-bilirubin to (ZE)-bilirubin, the most effective wavelength in vitro was 410 nm. For the structural photoisomerization, green light at 510 nm is the most efficient for causing cyclization of (ZZ)-bilirubin to (EZ)-cyclobilirubin via (EZ)-bilirubin and this may depend on a larger cross-section of (EZ)-bilirubin than (ZZ)-bilirubin in this spectral region and/or on a larger quantum yield for cyclization than geometric photoisomerization.

The effect of bilirubin photoisomers on unbound-bilirubin concentrations estimated by the peroxidase method.

Unbound bilirubin is oxidized to nearly colourless substances in the presence of H2O2 or ethyl hydroperoxide and horseradish peroxidase. To predict the risk of kernicterus (degenerated yellow pigmentation of nerve cells), this principle has been widely utilized for estimating the concentration of unbound bilirubin in hyperbilirubinaemic serum. However, the serum contains polar geometric photoisomers of bilirubin. Therefore, to clarify the effect of bilirubin photoisomer concentrations on unbound-bilirubin concentration, the concentration of bilirubin and its photoisomer and of unbound bilirubin in samples obtained from experiments in vivo and in vitro were simultaneously and individually estimated by h.p.l.c. and the peroxidase method. During photoirradiation, both in vivo and in vitro, the serum polar (ZE)-bilirubin IX alpha concentration increased remarkably, but unbound-bilirubin values were not affected at all. However, during experiments in vitro, unbound bilirubin concentrations increased only when concentrations of the rather polar (EZ)- and (EE)-cyclobilirubin IX alpha increased considerably in a human serum albumin-bilirubin solution irradiated with blue light. Thus it is concluded that unbound-bilirubin concentrations, and consequently the initial rate of the peroxidase reaction, is not accelerated by the increase in either (ZE)-bilirubin or (EZ)-cyclobilirubin concentration within the clinically observed range.

Metabolism of bilirubin and its photoisomers in newborn infants during phototherapy.

Bilirubin and its photoisomers in the biological fluids of a hyperbilirubinaemic newborn infant before and during phototherapy were analyzed by a recently improved HPLC method. In the serum, the percentages of (EZ)- and (ZE)-bilirubin in the total bilirubin concentration before phototherapy were approximately 10% and on average increased over 1.5-fold at 2 h after initiation of phototherapy. The percentage of the (EZ)-cyclobilirubin in the serum bilirubin was under 1%. In the bile, the mean concentration of (ZZ)-bilirubin, derived mainly from (ZE)-bilirubin, nearly tripled during phototherapy. The (EZ)-cyclobilirubin concentration in the bile was very low before phototherapy, increased nearly ten-fold at 3 h after initiation of phototherapy, and was 5- to 6-fold as high as that of (ZZ)-bilirubin. In the urine, upon exposure to light, the urinary concentration of (EZ)-cyclobilirubin is apparently equivalent to half of the biliary concentration of (ZZ)-bilirubin and one-fifth of that of (EZ)-cyclobilirubin. It was concluded that during phototherapy of neonatal hyperbilirubinaemia the structural photoisomer [(EZ)-cyclobilirubin] predominates considerably over the geometric photoisomer [(ZE)-bilirubin].