Controlled release of β-estradiol from biodegradable microparticles within a silicone matrix

Novel, biodegradable controlled release systems were prepared from biodegradable microparticles of poly(lactic acid-co-glycolic acid) containing β-estradiol in the presence or absence of silicone. The release behavior of β-estradiol from free microparticles as well as from microparticles embedded within a silicone matrix was compared with the release behavior shown by nonencapsulated β-estradiol within a silicone matrix. It was found that incorporating biodegradable microparticles within a silicone matrix lessens the initial burst of release often seen with these types of formulations and provides a controlled rate of drug release. In addition, the release rate of β-estradiol from biodegradable microparticles within silicone is higher than for unencapsulated β-estradiol in silicone. This type of formulation may be useful in a number of instances such as release of drugs from implants for which a simple drug-silicone formulation does not yield desired release behavior, formulations which are currently developed for microparticles but which may need to be removed if necessary, and implant formulations containing drugs which will not diffuse through silicone.     

Controlled release of TGF-β1 from a biodegradable matrix for bone regeneration

Although bone has a remarkable capacity for regenerative growth, there are many clinical situations in which the bony repair process is impaired. TGF-β₁ is a 25 kD homodimeric protein which modulates the growth and differentiation of many cell types. The ability of TGF-β₁, to promote bone formation suggests that it may have potential as a therapeutic agent in disease of bone loss. However, there still exists a need for an effective method of delivering TGF-β₁ to the site of an osseous defect for the promotion of bone healing. This paper describes a novel biodegradable controlled release system for TGF-β₁ comprised of poly (DL-lactic-co-glycolic acid) (PLPG) and demineralized bone matrix (DBM). The amount and activity of TGF-β_I released was determined using several methods including ¹²⁵I-labeled TGF-β₁ as a tracer, an enzyme linked immunosorbent assay (ELISA) and a growth inhibitory assay (GIA). Protein was released from the devices for time periods of more than 600 h. The amount of TGF-β₁ released was directly proportional to both the TGF-β₁ loading and the weight percent of DBM in the device. The release kinetics could be further controlled by applying polymeric coatings of varying porosity to the devices. The GIA indicated that between 80 and 90% of the TGF-β₁ released from the delivery system retained its bioactivity. The PLPG and DBM existed in phase separated domains within the device as determined by differential scanning calorimetry. Scanning electron microscopy suggested that the devices were sufficiently porous to allow bone ingrowth.     

Do extracellular matrix protein expressions change with cyclic reproductive hormones in pelvic connective tissue from women with stress urinary incontinence?

BACKGROUND: To evaluate differential expression of transforming growth factor (TGF-beta1), latent transforming factor-binding proteins (LTBP-1, LTBP-2) and elastin microfibril components (fibrillin-1 and fibrillin-2) in vaginal tissue from women with stress urinary incontinence (SUI). METHODS: In this case-control study, vaginal tissue from women in both phases of the menstrual cycle was obtained. Messenger RNA (mRNA) expressions of LTBP-1, LTBP-2, fibrillin-1, fibrillin-2 and TGF-beta1 were determined by relative real-time quantification PCR. Tissue localization was analysed by immunohistochemistry, and semiquantitative protein expression was evaluated by Western blot analysis. RESULTS: Vaginal wall fibroblasts synthesized all proteins tested. LTBP-1, LTBP-2 and TGF-beta1 co-localized with elastin microfibrils, fibrillin-1 and fibrillin-2 in the extracellular matrix. LTBP-1 mRNA and protein expressions were higher in control versus women affected with SUI in the proliferative phase (P = 0.04), while in the secretory phase, mRNA expression in cases was higher (P = 0.04). Fibrillin-1 mRNA was higher in women affected by SUI versus controls in both phases, but no statistical differences in fibrillin-1 protein expression were observed between the two groups in either phase. LTBP-2 and TGF-beta1 mRNA expressions showed the same trends as LTBP-1. CONCLUSION: LTBP-1, LTBP-2, TGF-beta1, fibrillin-1, and fibrillin-2 expressions are hormonally regulated in vaginal wall fibroblasts and differ in women affected by SUI when compared to controls. These data suggest a mechanism to regulate TGF-beta1 activity in pelvic connective tissue.     

A novel gene from Myxococcus xanthus that facilitates membrane translocation of an extracellular endoglucanase in Escherichia coli?

Expression in Escherichia coli of the Myxococcus xanthus gene celA, which encodes an extracellular endoglucanase, resulted in CelA being distributed between cytoplasm, periplasm and membrane. The presence of an adjacent open reading frame downstream from the full celA gene, or the absence of a putative lipoprotein signal sequence, confined CelA distribution to the periplasm and membrane, or to the cytoplasm and periplasm, respectively.     

Characterization of a novel α-amylase from Lipomyces kononenkoae and expression of its gene (LKA1) in Saccharomyces cerevisiae

A highly active -amylase (76 250 Da) secreted by the raw starch-degrading yeast Lipomyces kononenkoae strain IGC4052B was purified and characterized. Using high performance liquid chromatography (HPLC), end-product analysis indicated that the L. kononenkoae -amylase acted by endo-hydrolysis on glucose polymers containing -1,4 and -1,6 bonds, producing mainly maltose, maltotriose and maltotetraose. The following NH₂-terminal amino acids were determined for the purified enzyme: Asp-Cys-Thr-Thr-Val-Thr-Val-Leu-Ser-Ser-Pro-Glu-Ser-Val-Thr-Gly. The L. kononenkoae -amylase-encoding gene (LKA1), previously cloned as a cDNA fragment, was expressed in Saccharomyces cerevisiae under the control of the PGK1 promoter. The native signal sequence efficiently directed the secretion of the glycosylated protein in S. cerevisiae. De-glycosylation of the enzyme indicated that post-translational glycosylation is different in S. cerevisiae from that in L. kononenkoae. Zymogram analysis indicated that glycosylation of the protein in S. cerevisiae had a negative effect on enzyme activity. Southern-blot analysis revealed that there is only a single LKA1 gene present in the genome of L. kononenkoae.     

Characterization and localization of α-connectin (titin 1): An elastic protein isolated from rabbit skeletal muscle

Summary A simplified procedure to isolate-connectin (titin 1, TI), a gigantic elastic protein, from rabbit skeletal muscle is described. A rapid column chromatography step to concentrate-connectin is introduced. Separation of-connectin from-connectin is introduced. Separation of-connectin from-connectin (titin 2, TII) in the presence of 4 M urea at pH 7.0 did not cause any change in the secondary structure of-connectin as judged by circular dichroic spectra. Ultraviolet absorption spectra and the amino acid composition of-connectin (MW, approximately 3×10⁶) were similar to those of its proteolytic product,-connectin (MW, approximately 2×10⁶). Circular dichroic spectra suggested that both- and-connectin consist of 60%-sheet and 30%-turn. It thus appears that the whole elastic filament of connectin has a folded-strand structure. Proteolysis of-connectin by calpain resulted in formation of-connectin and smaller peptides. The-connectin interacted with both myosin and actin filaments similarly to-connectin. Polyclonal antibodies raised against 1200 kDa peptides obtained from aged rabbit skeletal myofibrils reacted with-connectin (titin 1, TI) but only weakly with-connectin (titin 2, TII) in rabbit skeletal muscle. Immunoelectron microscopy and indirect immunofluorescence microscopy revealed that the antibodies bound at the Z-line and at the epitope regions in the I-band near the binding site of a monoclonal antibody SMI whose position depends on sarcomere length. It thus appears that-connectin extends from the edge of M-line to the above epitope region in the I-band.     

The new HLA allele, HLA-A*03:57, differs from HLA-A*03:01 by two amino acids at positions 76 and 77 in the α2 domain affecting the pocket F of the peptide-binding groove

HLA-A*03:57 has two amino acid exchanges in exon 2 at adjacent positions of the peptide-binding groove.     

In vitro and in vivo α-amylase and α-glucosidase inhibiting activities of the protein extracts from two varieties of bitter gourd (<Emphasis Type="Italic">Momordica charantia</Emphasis> L.)

Background

α-amylase and α-glucosidase digest the carbohydrates and increase the postprandial glucose level in diabetic patients. Inhibiting the activity of these two enzymes can control postprandial hyperglycemia, and reduce the risk of developing diabetes. Bitter gourd or balsam pear is one of the important medicinal plants used for controlling postprandial hyperglycemia in diabetes patients. However, there is limited information available on the presence of α-amylase and α-glucosidase inhibiting compounds. In the current study, the protein extracts from the fruits of M. charantia var. charantia (MCC) and M. charantia var. muricata (MCM) were tested for α-amylase and α-glucosidase inhibiting activities in vitro, and glucose lowering activity after oral administration in vivo.

Results

The protein extract from both MCC and MCM inhibited the activity of α-amylase and α-glucosidase through competitive inhibition, which was on par with Acarbose as indicated by in vitro percentage of inhibition (66 to 69 %) and IC50 (0.26 to 0.29 mg/ml). Both the protein extracts significantly reduced peak blood glucose and area under the curve in Streptozotocin-induced diabetic rats, which were orally challenged with starch and sucrose.

Conclusions

Protein extracts from the fruits of the two varieties of bitter gourd inhibited α-amylase and α-glucosidase in vitro and lowered the blood glucose level in vivo on par with Acarbose when orally administrated to Streptozotocin-induced diabetic rats. Further studies on mechanism of action and methods of safe and biologically active delivery will help to develop an anti-diabetic oral protein drug from these plants.

     

A novel amino acid substitution at the receptor-binding site on the hemagglutinin of H3N2 influenza A viruses isolated from 6 cases with acute encephalopathy during the 1997–1998 season in Tokyo

Summary.  We analyzed the hemagglutinin (HA) genes of influenza A viruses consisting of 6 strains isolated from cerebrospinal fluids of patients with encephalopathy and 3 isolates from throat washes of patients without central nervous system symptoms during the 1997–1998 season in Tokyo. Aligned 9 amino acid sequences showed 7 common substitutions compared with A/Wuhan/359/95 (vaccine strain used in the season in Japan), which were allocated to three different antigenic sites on the H3 HA molecule. It is noted that a novel substitution at the receptor-binding site (Tyr-137 to Phe) was found exclusively in the isolates from the patients with encephalopathy. Received May 29, 1998 Accepted September 3, 1998