Effect of a lysyl hydroxylase inhibitor,minoxidil, on ultrastructure and behavior of cultured rabbit subconjunctival fibroblasts

Background: Minoxidil is an inhibitor of lysyl hydroxylase, an enzyme involved in collagen production, and decreases collagen production in vitro. We investigated the in vitro effects of minoxidil on behavior such as proliferation and migration of rabbit subconjunctival fibroblasts (SCFs). The ultrastructural effect of the drug on SCFs was also examined. Methods: Proliferation of SCFs and closure of the defect produced in monolayer cultures in the presence or absence of minoxidil was studied. The ultrastructure of SCFs treated with minoxidil was also examined. Results: Minoxidil inhibited SCF proliferation and the closure of the defect produced in monolayer cell sheets. Ultrastructural observations revealed extensive areas of irregularly dilated endoplasmic reticulum in cells treated with minoxidil, indicating the accumulation of protein, probably underhydroxylated collagen precursors, in the cisternae of endoplasmic reticulum. Conclusions: The results indicated that minoxidil attenuated cellular activities of SCFs such as proliferation and migration in vitro. The exact mechanism of the inhibitory effects of minoxidil on these cellular activities is unknown. The findings suggest that the drug might help to prevent bleb scarring after glaucoma filtering surgery.     

Immunolocalization of prolyl 4-hydroxylase in fibroblasts cultured from Tenon's capsule of humans

Background: The excessive accumulation of extracellular matrix (ECM) with the repopulation of fibroblasts may lead to an unsuccessful outcome of glaucoma filtering surgery. We examined the immunolocalization of ECM components and prolyl 4-hydroxylase, an enzyme involved in collagen biosynthesis, in cultured Tenon's capsule fibroblasts (TCFs) of humans to evaluate the production of ECM in the cells. Methods: We used light microscopy to evaluate the immunolocalization of prolyl 4-hydroxylase and ECM components, collagen types I, III, and IV, cellular fibronectin, and laminin in TCFs. Ultrastructural localization of the enzyme was also evaluated by electron microscopy. Results: Immunoreactivity with monoclonal antibodies against the and subunits of the enzyme or with the polyclonal antibody against it was detected in the cytoplasm of the cells in a fine granular pattern, indicating its localization in the indoplasmic reticulum (ER). Immunoreactivity for the enzyme was detected in the cisternae of the ER on electron microscopy. Types I and III collagen reactivities were also observed in the cytoplasm in a fine granular pattern. T reactivity was present diffusely on the cell surface. The distribution of laminin reactivity in the cytoplasm resembled that of types I and III collagen. Cellular fibronectin reactivity was observed in the ECM in a reticular pattern. Conclusion: Prolyl 4-hydroxylase was located in the cisternae of the ER. TCFs produced a variety of ECM components in vitro. The results provide insight into the fibrotic process during scar formation at the site of a bleb following filtering surgery.     

γ-Glutamyltransferase test as a new tool for identification of seminal stains

Summary A simple qualitative method for identification of seminal stains based on a high activity of -glutamyltransferase (-GTP) in human semen is described. It employs the release of -naphthylamine from N--glutamyl--naphthylamide by the -GTP action; -naphthylamine couples with Fast Garnet GBC salt to produce a strong brownish-red color. The data on its simplicity, specificity, and stability show that the present method is suitable for medicolegal examination of seminal stains as a preliminary test.     

Serum fluoride as an indicator of occupational hydrofluoric acid exposure

Summary To define the relationship between ionic fluoride concentration in the serum of workers and the amount of hydrofluoric acid (HF) in the work environment, pre-and postshift serum and urine samples of 142 HF workers and 270 unexposed workers were examined. The maximum and minimum concentrations of HF in the air in each workshop varied from the mean by less than 30%. The pre-exposure levels of serum and urinary fluoride in HF workers were higher (P < 0.001) than the control values. This suggests that fluoride excretion from the body continues for at least 12 h. The postshift serum and urinary fluoride concentrations of these workers were significantly higher (P < 0.001) than the preshift concentrations. A good correlation (r = 0.64) was obtained between postshift serum fluoride and postshift urine fluoride. There was a linear relationship between mean serum fluoride concentration and HF concentration in the workshop. A mean fluoride concentration of 82.3 g/l with a lower fiducial limit (95%, P = 0.05) of 57.9 g/l was estimated to correspond to an atmospheric HF concentration of 3 ppm. This is the maximum allowable environmental concentration recommended by the Japanese Association of Industrial Health, and it is also the threshold limit value suggested by the American Conference of Governmental Industrial Hygienists. The results demonstrate that exposure to HF can be monitored by determining the serum fluoride concentration.     

Prognostic significance of clinical and angiogenesis-related factors in low-grade oligodendrogliomas

BACKGROUND: Keeping in mind that oligodendrogliomas have unpredictable biological behavior, the aim of this study is to investigate the prognostic significance of VEGF expression and microvessel density in a homogeneous series of low-grade oligodendrogliomas. METHODS: For this study 36 patients with a low-grade oligodendroglioma treated by surgical resection and radiotherapy were selected. At the time of surgery, in all cases the Karnofsky Performance Scale (KPS) score was more than 70, and the study of the resected tumor disclosed a Ki-67/MIB-1 labeling index (MBI-1 LI) less than 1%. In this homogeneous series, immunohistochemical studies were performed using monoclonal antibodies against VEGF in order to study the expression of this cytokine, and against vascular endothelial CD-34 antigen, in order to identify microvessels. RESULTS: Our results show that in contrast to low-grade astrocytomas, low-grade oligodendrogliomas lacked immunoreactive VEGF. Oligodendrogliomas with low vascular density (less than 20 microvessels per microscopical field, at 200 x) or high vascular density (more than 100 microvessels per field, at 200 x) were identified, but this factor had no influence on the survival rate of patients. On the other hand, analysis of the present series showed that clinical factors, such as age or extent of surgical resection, were not significantly associated with survival. CONCLUSIONS: In contrast to low-grade astrocytomas, the angiogenesis score of low-grade oligodendrogliomas (counting the number of microvessels in tumor tissue) adds little information to help predict tumor behavior.     

New‐onset fulminant type 1 diabetes after severe acute respiratory syndrome coronavirus 2 vaccination: A case report

Fulminant type 1 diabetes is characterized by a rapid progression of insulin deficiency triggered by viral infection. Here, we report a case of a 45‐year‐old Japanese woman with fulminant type 1 diabetes that developed 8 days after receiving messenger ribonucleic acid vaccine against severe acute respiratory syndrome coronavirus 2. She had been healthy and had no symptoms suggestive of viral infection before the vaccination. Laboratory tests showed exhaustion of insulin secretion and negative results for islet autoantibodies. Human leukocyte antigen genotype analysis showed the DRB1*04:05 and DQB1*04:01 alleles. This is the first case report of new‐onset fulminant type 1 diabetes after severe acute respiratory syndrome coronavirus 2 vaccination, and suggests that a severe acute respiratory syndrome coronavirus 2 vaccine might trigger the onset of fulminant type 1 diabetes in susceptible individuals. However, a causal relationship remains to be identified, and further studies are required to determine the incidence of such cases.     

Reduction of oocyte lipid droplets and meiotic failure due to biotin deficiency was not rescued by restoring the biotin nutritional status

BACKGROUND/OBJECTIVESOocyte lipid droplets play a crucial role in meiosis and embryo development. Biotin is associated with fatty acid synthesis and is the coenzyme for acetyl-CoA carboxylase (ACC). The effects of a biotin deficiency on the oocyte lipid metabolism remain unknown. This study examined the effects of a biotin deficiency and its replenishment on murine 1) oocyte lipid droplet levels, 2) ovary lipid metabolism, and 3) oocyte meiosis.MATERIALS/METHODSMice were divided into 3 groups: control, biotin deficient (BD), and recovery groups. The control and BD groups were fed a control diet or BD diet (0.004 or 0 g biotin/kg), respectively. The recovery group mice were fed a BD diet until day 21, and were then fed the control diet from days 22 to 64. This study then quantified the oocyte lipid droplet levels, assessed the oocyte mitochondrial function, and examined the ability of oocytes to undergo meiosis. Ovarian phosphorylated ACC (p-ACC), lipogenesis, β-oxidation, and ATP production-related genes were evaluated.RESULTSThe BD group showed a decrease in lipid droplets and mitochondrial membrane potential and increased p-ACC levels. In the recovery group, the hepatic biotin concentration, ovarian p-ACC levels, and mitochondrial membrane potential were restored to the control group levels. On the other hand, the quantity of lipid droplets in the recovery group was not restored to the control levels. Furthermore, the percentage of oocytes with meiotic abnormalities was higher in the recovery group than in the control group.CONCLUSIONSA biotin deficiency reduced the oocyte lipid droplet levels by downregulating lipogenesis. The decreased lipid droplets and increased oocyte meiosis failure were not fully restored, even though the biotin nutrition status and gene expression of lipid metabolism was resumed. These results suggest that a biotin deficiency remains robust and can be long-lasting. Biotin might play a crucial role in maintaining the oocyte quality.     

Protective effect of alpha-lipoic acid in methotrexate-induced ovarian oxidative injury and decreased ovarian reserve in rats

To determine whether the possible oxidative effect of methotrexate (Mtx) on ovary and to evaluate the effectiveness of alpha lipoic acid (ALA), which may be useful in many oxidative stress models.

Thirty-two female Wistar-albino rats were randomly divided into four groups; control group, alpha lipoic acid group (ALA 100?mg/kg, 10?days), multiple dose Mtx group (Mtx 1?mg/kg 1, 3, 5, 7?days) and Mtx and ALA group (Mtx 1?mg/kg 1, 3, 5, 7?days and ALA 100?mg/kg, 10?days). Serum total antioxidant status (TAS), total oxidant status (TOS) and oxidative stress index (OSI), tumor necrosis factor-alpha (TNF-α), tissue malondialdehyde (MDA) and activities of glutathione peroxidase (GSH-Px) and catalase (CAT) and anti-Mullerian hormone (AMH) and total ovarian follicle count were evaluated.

Mtx administration caused a significant decrease in TAS, a significant increase in TOS and OSI, a significant increase in MDA levels and a decrease in GSH-Px and CAT activity. Moreover the proinflammatory cytokine (TNF-α) was increased in the Mtx group. And AMH values and total follicle count were significantly decreased in Mtx group. However, ALA treatment reversed biochemical results and AMH levels and total follicle count.

Alpha lipoic acid ameliorates methotrexate induced oxidative damage of ovarian in rats.     

Visualization of the photodegradation of a therapeutic drug by chemometric-assisted fluorescence spectroscopy

The fluorescence spectral fingerprint, also known as the excitation-emission matrix (EEM), is used to assess and visualize therapeutic drug photodegradation in combination with chemometrics. Examination of EEM-parallel factor analysis (PARAFAC) data showed that an individual component was easily separated from a mixture of photogenerated products of a heterocyclic pharmacophore, in this case, phenothiazine drugs (PTZs). Detailed investigations of both structure–EEM relationships and kinetics revealed that the components extracted from EEM–PARAFAC could be quantitatively attributed to such photogenerated products as phenothiazine sulfoxide and carbazole derivatives. EEM in combination with principal component analysis (PCA) could be used as a mapping tool to visualize information of the photodegradation process of PTZs. We also assessed the photostability of various types of PTZs containing side chains by using validated EEM–PARAFAC methodology.

Drug quality and assurance changes with time under the influence of a variety of environmental factors, such as light, temperature, and moisture.     

Pharmacodynamic evaluation of meropenem,cefepime, or aztreonam combined with a novel β-lactamase inhibitor,nacubactam, against carbapenem-resistant and/or carbapenemase-producing Klebsiella pneumoniae and Escherichia coli using a murine thigh-infection model

BackgroundCarbapenem-resistant Enterobacterales (CRE) and carbapenemase-producing Enterobacterales (CPE) are difficult to treat and are a serious public health threat. Nacubactam (NAC) is a novel non-β-lactam diazabicyclooctane β-lactamase inhibitor with in vitro activity against some Enterobacterales expressing classes of β-lactamases.MethodsThe antimicrobial efficacy of meropenem (MEM), cefepime (FEP), and aztreonam (ATM), each in combination with NAC, were assessed in vitro and in vivo against Klebsiella pneumoniae and Escherichia coli. Ten isolates, including CRE and/or CPE with β-lactamase genes, were used in this study. The relationship between phenotype and in vivo efficacy was assessed in a murine neutropenic thigh-infection model. Efficacy was determined by the change in bacterial quantity.ResultsThe results of the in vitro study showed the minimum inhibitory concentrations of the combination of NAC with either MEM, FEP, or ATM in a 1:1 ratio were 2 to >128-fold lower than those of MEM, FEP, or ATM alone against CRE+ isolates. In addition, combinations of β-lactams and NAC administered in the murine thigh-infection model showed greater efficacy against CRE+/CPE+, CRE+/CPE-, and CRE-/CPE+ isolates harboring various β-lactamase genes (IMP-1, IMP-6, KPC, DHA-1, or OXA-48) compared with MEM, FEP, ATM, and NAC alone.ConclusionMEM, FEP, or ATM in combination with NAC showed potent in vivo antimicrobial activity in a murine thigh-infection model caused by K. pneumoniae and E. coli, including CRE and/or CPE isolates. These findings indicate that these combinations of β-lactams and NAC are potential candidates for the treatment of CRE and/or CPE infections.