[Genotype-phenotype analysis of Fabry disease caused by GLA gene variation in a pedigree].

Objective: To investigate the clinical phenotype and genetic characteristics of patients with Fabry disease caused by a GLA variant, IVS4+919G>A. Methods: It was a prospective study. Fabry disease screening was conducted among high-risk population in Ninghai from October 2021 to August 2023. Those children with decreased α-galactosidase enzyme activity<2.40 µmol/(L·h) or elavated Lyso-GL-3 level>1.10 µg/L in dried blood spot (DBS) method underwent GLA genetic testing for diagnosis confirmation. Meanwhile, family screening was carried out. A proband and his family members diagnosed with Fabry disease were research subjects. The clinical and genetic characteristics of patients with Fabry disease caused by the GLA variant (IVS4+919G>A) were analyzed. Results: The female proband aged 9.8 years with pain in both lower limbs as the initial symptom was found to have a heterozygous GLA variant IVS4+919G>A among 102 patients. In family screening, there were 4 family members (proband's father, elder sister, elder male cousin and elder female cousin) with Fabry disease and a family member (proband's fifth aunt) with a GLA variant. Among these 4 diagnosed family members, the elder male cousin of the proband, a boy aged 13.2 years had a heterozygous GLA variant, IVS4+919G>A with intermittent pain in both lower limbs as the initial symptom. The proband's father had knee joint pain. The proband's elder sister had decreased vision and his elder female cousin had no obvious symptoms. The proband's fifth aunt with a GLA variant had decreased vision. Conclusions: High-risk screening in children and family screening are helpful for early diagnosis and treatment of Fabry disease. Neuropathic pain may be a early symptom in children with Fabry disease caused by the GLA variant, IVS4+919G>A.

Role of adiponectin in adipose tissue wound healing.

The purpose of this study was to investigate the mechanism behind adipose tissue wound healing (ATWH). The preadipocyte cell line 3T3-L1 was cultured and expression of adiponectin receptors (AdipoR1/2) was detected by immunohistochemistry and reverse transcription polymerase chain reaction. The concentration of adiponectin secreted at different cell densities was measured by enzyme-linked immunosorbent assay, while preadipocyte proliferation and migration were determined in vitro by MTT and wound closure assays. AdipoR1/2 were found to be expressed in 3T3-L1 preadipocytes. There were no statistically significant differences in the concentrations of adiponectin secreted by cell solutions of different densities (P > 0.05). In addition, adiponectin was seen to promote the growth and migration of preadipocytes. In conclusion, adiponectin may regulate ATWH by promoting preadipocyte proliferation and migration, and its systemic and/or local application is proposed as a promising therapeutic approach for the treatment of wounds incurred as a result of surgery.

Ulex europaeus agglutinin-I binds to developing gastrin cells.

We have previously reported that antropyloric gastrin (G) and somatostatin (D) cells derive from precursor (G/D) cells that coexpress both hormones. We have now analyzed this endocrine cell pedigree for binding of Ulex europaeus agglutinin-I (UEA-I), which previously has been reported to represent a useful marker for cell differentiation. Subpopulations of G/D, D, and G cells were all found to express UEA-I binding. Labelling with bromodeoxyuridine showed that UEA-I positive G cells possessed a higher labelling index than UEA-I negative G cells. These data suggest that the UEA-I positive G cells represent maturing cells still involved in DNA synthesis and cell division. Electron microscopically, specific UEA-I binding sites were localized to the secretory granules and the apical cell membrane of G cells. We conclude that UEA-I represents a differentiation marker for G cells. Moreover, the presence of UEA-I binding sites in these cells may be relevant for Helicobacter pylori-mediated disturbances of gastric acid secretion and gastrin hypersecretion.

[In vivo and in vitro metabolism of tricyclopinate hydrochloride in the rat].

The metabolism of tricyclopinate hydrochloride(TCPN), a new anticholinergic agent, was studied by in situ perfused rat liver preparation. Two metabolites were isolated by HPLC. Metabolite I was identified as an N-demethylation product, while metabolite II was shown to be an arylhydroxylation product. Metabolite I and metabolite II were also isolated from the urine of the rat given TCPN i.g. This result shows that the two metabolites from the perfused rat liver are just the final metabolites produced in vivo. The metabolites in rat liver microsomes were also identified as those just described. These results suggest that the biotransformation reaction of TCPN is mainly catalyzed by the enzyme in rat liver microsomes. The results of experiment on receptor activity indicate that the action potency with mAChR of metabolite I and metabolite II was only 1/20 and 1/50, respectively, of that of TCPN.

[Pharmacokinetics of naltrexone hydrochloride and naltrexone glucuronide in the dog].

Pharmacokinetics of naltrexone hydrochloride (NTX) and naltrexone glucuronide was studied in the dog using HPLC-electrochemical detection with naloxone as internal standard. After iv 5 mg or po 10 mg NTX, the plasma concentration-time curves of NTX were found to fit to a two-compartment model and a single compartment with first-order absorption. The elimination half-lives of NTX were 78 +/- 6 min and 74 +/- 6 min, respectively. Although NTX could be absorbed rapidly in the dog after po administration, the plasma concentration of the parent drug was very low and its absolute bioavailability was 15.8%. The experiments showed that the major metabolite of NTX in dog plasma was beta-glucuronidase-hydrolyzable conjugate. Dosing NTX intravenously and orally, the plasma levels of the conjugate were 1.3 and 23 times as high as that of the parent drug, the elimination half-lives of the glucuronide from plasma were 3.4 h and 12.6 h, respectively. The results indicate that NTX is subjected to a marked first-pass effect in the dog after oral administration.

Noninvasive method for monitoring ethanol in fermentation processes using fiber-optic near-infrared spectroscopy.

Short-wavelength near-infrared (SW-near-IR) spectroscopy (700-1100 nm) is used for the determination of ethanol during the time course of a fermentation. Measurements are performed noninvasively by means of a photodiode array spectrometer equipped with a fiber-optic probe placed on the outside of the glass-wall fermentation vessel. Pure ethanol/water and ethanol/yeast/water mixtures are studied to establish the spectral features that characterize ethanol and to show that determination of ethanol is independent of the yeast concentration. Analysis of the second-derivative data is accomplished with multilinear regression (MLR). The standard error of prediction (SEP) of ethanol in ethanol/water solutions is approximately 0.2% over a range of 0-15%; the SEP of ethanol in ethanol/yeast/water solutions is 0.27% (w/w). Results from the mixture experiments are then applied to actual yeast fermentations of glucose to ethanol. By use of a gas chromatographic method for validation, a good correlation is found between the intensity of backscattered light at 905 nm and the actual ethanol. Additional experiments show that a calibration model created for one fermentation can be used to predict ethanol production during the time course of others with a prediction error of 0.4%.

[Immunohistochemical study of thymus in patients with congenital heart disease or rheumatic heart disease].

A series monoclonal antibodies were used to study the intrathymic distribution of T and B cells and the process of intrathymic differentiation of T cells on frozen sections of 14 patients' thymuses by a more sensitive immunohistochemical method. The observation indicated that most of the cortical lymphocytes reacted with Leu 1, Leu 2 a, Leu 3 a and Leu 4 antibodies, thus indicating coexpression of multiple antigen on cortical lymphocytes. In cortex, the numbers of Leu 1 and Leu 4 positive cells were less than medulla. In contrary, the numbers of Leu 2 a and Leu 3 a positive cells in the cortex were more than the medulla: and the medullary thymocytes showed segregation into Leu 2 a+ Leu 3 a- and Leu 3 a+ Leu 2 a- subsets. The ANAE+ and AIG 3+ cells were almost limited to the medulla. Hence, the staining profile of medulla approximated the staining pattern of peripheral T cell subsets, which were programmed for their respective role as helper and suppressor cells. Our results supported the conclusion that the mature T cells presented in the medulla were derived from the immature cells in the cortex. In addition, the expression of Leu 7 was not only limited to the NK cells scattered in the medulla, but also found at the cortical-septa border. On the other hand, a small number of B lymphocytes scattered in the medulla stained with Leu 14, OKB-2, BA-1 and IgM antibodies. The Hassall's corpuscles were cross reaction with the B cell antibodies mentioned above; while the epithelial cells in thymic parenchyma were only stained with IgM antibody. Based on these findings we conclude that the human thymus predominantly consist of T cells, but it also contains a fraction of B cells and epithelial cells.

Comparative distribution of carbonic anhydrase isozymes III and II in rodent tissues.

Carbonic anhydrase (CA) III was demonstrated immunocytochemically in epithelium in some regions of salivary gland ducts, colon, bronchi, and male genital tract and in adipocytes, in addition to skeletal muscle and liver where the isozyme was previously localized. Basal cells beneath the submandibular gland's excretory ducts in guinea pig stained for CA III. Carbonic anhydrase III occurred alone in some and with CA II in other sites but was often absent from CA-II-containing types of cells. This was exemplified by CA III's abundance in CA-II-positive proximal colon and its sparsity in the CA-II-rich distal colon of the mouse. Striated ducts in guinea pig, but not mouse salivary glands, stained darker for CA and appeared accordingly to function more actively in ion transport compared with excretory ducts. Carbonic anhydrase content varied among genera in liver and pancreas and between mouse species and strains in salivary glands and kidney. Newly observed murine sites of CA II activity included Auerbach's plexus and a population of leukocytes infiltrating the lamina propria in small intestine, and several types of cells in the male genital tract. In immunoblot tests, antisera to CA III showed no cross reactivity with antisera to CA II, but those to CA II disclosed weak cross reactivity with CA III.

[Analysis of musk rheumatic oil by GC/FTIR].

This paper studied musk rheumatic oil by GC/FTIR. Ten main components were identified qualitatively, and methyl salicylate, eugenol, cinnamaldehyde were determined by GC internal standard method quantitatively. Average recoveries are within 100 +/- 1.0%, variation coefficients are less than 2.0%.

[Effects of substance P on the contractile response of longitudinal muscle of rat ileum to 5-HT].

The purpose of this study was to observe the effects of substance P (SP) on the contractile response of longitudinal muscle with myenteric plexus (LM-MP) of rat ileum induced by 5-HT in vitro and to analyse its mechanism. The results were as follows: (1) Subthreshold dose of SP (5 nmol/L) potentiated the 5-HT-induced contractile response; (2) Both [D-Pro2 D-Trp7,9]-SP and atropine inhibited or blocked this potentiated effect suggesting that the effect was mediated by SP receptors; (3) This effect was not blocked by bromolysergic acid diethylamide (BOL). The results suggest that SP of subthreshold dose might modulate the effect of 5-HT, and a cholinergic mechanism might be involved in the modulation.

[A study of lymphocyte subsets in human normal tonsil and lymph node].

A series of T and B lymphocyte specific monoclonal antibodies was used to determine the localization of lymphocyte subpopulation in frozen and paraffin tissue sections of human normal tonsil and lymph node by means of immunocytochemical technique. In the paracortical and interfollicular area of tonsil and lymph node, most lymphocytes reacted with Leu 1, Leu 3 a, Leu 4 and OKT4. The numbers of Leu 2 a and OKT8 positive cells were rare in tissue. These cells were not only limited in paracortical area, they also appeared in considerable numbers in medullary cords of lymph nodes. Leu 2 a and OKT 8 positive cells decreased with prominent follicular hyperplasia of tonsils. In addition, substantial leu 3 a and Leu 4 cells were found in the germinal centers. This finding supports the importance of these lymphocyte subsets in regulation of human immune response. In the mantle zone of secondary follicles, the majority of lymphocytes were positive for OKB 2 and BA 1, whereas, the IgM positive cells were predominately observed in the cytoplasma and extracellular substance of B lymphocytes in the germinal centers, but the lymphocytes bearing sIgM were rarely observed. In the mantle zone, the IgM were frequently found on the surface of membrane of small lymphocytes, however, the staining intensity was much than that in the germinal centers.

Immunocytochemistry of ion transport mediators in the genital tract of female rodents.

With immunocytochemistry, we have determined distribution of sodium, potassium-adenosine triphosphatase (Na+, K+-ATPase) and of three isoenzymes of carbonic anhydrase (CA) and have shown absence of the chloride channel, Band 3 protein, in the genital tract of female rodents. Staining for Na+,K+-ATPase was strongest in the ampullary oviduct and uterine glands in the mouse. In the mouse and rat ovary, immunostaining evidenced CA I, II, and III in theca interna cells where the enzyme could affect the pH of follicular fluid. The zona pellucida of the ovary and cytoplasmic foci in follicular granulosa cells stained for content of only CA I in mouse ovary, suggesting synthesis of a zona pellucida component by granulosa cells. CA II in mouse oviductal epithelium increased from the negative infundibulum to the variably positive ampulla and isthmus to the uniformly positive interstitial segment. The content of CA III varied inversely to that of CA II. The prevalence of CA II-positive cells apparently corresponded with that of nonciliated cells, whereas abundance of CA III-positive cells concurred with that of ciliated cells in regions of the mouse oviduct. The rat oviduct lacked CA II but, like that of the mouse, showed CA III in the proximal region. The staining for CA II in surface epithelium exceeded the reactivity in glandular epithelium in the mouse uterus, except during estrus. In contrast, rat uterus evidenced CA II in glandular but not surface epithelium. These results testify to possible significance of various ion transport mechanisms for biologic activities of diverse cells in the female genital tract.

Evidence for the blood-urine barrier depending on urothelium and carbonic anhydrase positive fibroblasts.

A subepithelial multilayer of abundant fusiform cells has been distinguished cytochemically in the urinary bladder and ureter in mice and rats. These distinctive cells stained selectively for carbonic anhydrase (CA) isozymes I and III. Immunonegativity for keratin and Na+,K+-ATPase differentiated the CA-positive cells from epithelial cells and their lack of immunoreactivity for actin distinguished them from smooth muscle cells. Immunostaining for vimentin, blue staining with the trichrome method, location in an exceptionally dense collagen stroma, and ultrastructural appearance related the multilayer cells to fibroblasts. A loosely collagenous, less cellular lamina propria separated the CA-positive suburothelial zone from the smooth muscle wall in the rodent urinary bladder. Ureter lacked the loose lamina propria, and the presence of such a collageneous layer in bladder therefore correlated with distensability of the organ. The presence of CA uniquely in the fibroblastoid cells applied intimately to ureter and bladder epithelium implies a specialized function of these cells, possibly one concerned with the barrier between blood and hypertonic urine. Cytochemical demonstration of keratin and fucose-rich glycoconjugate in the plasmalemma of superficial urothelial cells indicates a role for these components in passively maintaining the blood-urine barrier. The observed distribution of Na+,K+-ATPase in mid and deep urothelial cells implicates this enzyme and these cells in actively maintaining the urine's hypertonicity. Basal urothelial cells contained glycoconjugate with terminal galactose in their plasmalemma. Ultrastructural features suggesting involution of superficial urothelial cells further evidence restriction of active ion transport to the deeper cells.

Lectin detection of renal glycogen in rats with short-term streptozotocin-diabetes.

Earlier histochemical findings from our laboratory have shown that a lectin (agglutinin) from Griffonia simplicifolia, which reportedly binds to terminal N-acetylglucosamine residues in glycoconjugate oligosaccharides also shows affinity for glycogen. In the present study, the lectin was conjugated to horseradish peroxidase and applied to paraffin sections of kidney from streptozotocin-diabetic rats, insulin-treated and untreated, and age-matched control rats. Griffonia simplicifolia agglutinin II detected glycogen in cortical ascending thick limbs of untreated diabetic rat kidneys as early as 24 h following injection of streptozotocin. The number of stained cells increased steadily so that by day 14 of diabetes the lectin reacted with nearly all of the cells lining ascending thick limbs in the cortex and adjacent outer stripe of the outer medulla. Glycogen was never identified in the inner medullary stripe. Comparison of Griffonia simplicifolia agglutinin II and periodic acid-Schiff staining revealed that periodic acid-Schiff could not clearly detect glycogen until 14 days following injection of streptozotocin, which substantiated earlier claims that Griffonia simplicifolia agglutinin II might be a more sensitive indicator of glycogen than periodic acid-Schiff. The distribution of glycoconjugate containing terminal N-acetylglucosamine stainable with the lectin was unchanged in diabetic kidneys. Griffonia simplicifolia agglutinin II served in the present study to further characterise the sequence of abnormal glycogen accumulation in streptozotocin-diabetic rat kidneys. In addition, it was shown that the lectin's ability to antedate periodic acid-Schiff detection of glycogen has utility in histochemical investigations in diabetes.

Biochemical and ultrastructural study of Leu M1 antigen in Reed-Sternberg cells: comparison with granulocytes and interdigitating reticulum cells.

A group of monoclonal antibodies was shown to react with glycoconjugates containing a sugar sequence--lacto-N-fucopentaose III (LNF-III)--in granulocytes and in some normal nonlymphoid cells. The antibodies including anti-Leu M1, anti-My-1, WGHS 29-1, 534F-8, and 538F-12 of the immunoglobulin M-type were used to study the biochemical properties of LNF-III antigens in granulocytes, interdigitating reticulum cells, and neoplastic cells of Hodgkin's disease. In contrast to the presence of an abundant LNF-III glycolipid in granulocytes, the Hodgkin's neoplastic cells had no LNF-III glycolipid or contained only minimal amounts; however, both LNF-III glycoconjugates isolated from Hodgkin's neoplastic cells and interdigitating reticulum cells appeared to be a similar, if not an identical, 150,000-molecular-weight glycoprotein. The neoplastic cells in Hodgkin's disease appeared to show a biochemical property more closely related to interdigitating reticulum cells than any other cells in the monocyte-granulocyte-histiocyte system.