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PubMed1976

Synthesis and properties of carbonylbis(methionyl)insulin, a proinsulin analogue which is convertible to insulin by cyanogen bromide cleavage.

The preparation and use of carbonylbis (L-methionine p-nitrophenyl ester) as a reversible cross-linking reagent for insulin are described. The reaction of 1 equiv of reagent with zinc insulin in dimethylformamide in the presence of triethylamine yields as one of the products NalphaA1, NepsilonB29-carbonylbis(methionyl)insulin, (CBM-insulin). The CBM-insulin was characterized by end group analysis and by the products formed on tryptic and chymotryptic cleavage. It possessed 91% of the immunological and 6.5% of the hormonal activity of insulin. Treatment of CBM-insulin with cyanogen bromide (CNBr) in 70% formic acid for 1 h resulted in nearly complete removal of the methionine bridge to yield insulin. A small amount of a side product was removed on DEAE-cellulose at pH 7.2 to give an overall recovery of insulin of 70-80%. Oxidative sulfitolyses of CBM-insulin gave the hexa(S-sulfonate) which was reduced with dithiothreitol to yield reduced CBM-insulin. The latter compound, containing 6 sulfhydryls, exhibited a pH-dependent circular dichroic spectrum. The form at pH 10 exhibited a spectrum typical of random coil which was converted to a form at pH 7.8 which was characterized by a negative extremum at 213 nm. The change in the spectrum at 213 nm with pH was characterized by an apparent pKa of 8.5. Studies on the reoxidation of reduced CBM-insulin were performed at pH values between 7.8 and 10 and at protein concentrations of 0.01-1 mg/ml. The best yields (ca. 85%) of the correctly paired disulfide bonds were obtained in reoxidations at pH 9.5-10 at protein concentration of 0.01-0.1 mg/ml. CBM-insulin, which had been isolated from reoxidation at high pH of the reduced CBM-insulin, was cleaved by CNBr to yield a fully active insulin in an overall yield of 60% from the reduced CBM-insulin.

Adipose Tissue↗

PubMed1977

The human cutaneous basement membrane--anchoring fibril complex: preparation and ultrastructure.

The human cutaneous basement membrane (CBM) has been isolated together with the anchoring fibrils (AF) of the dermis. These structures are removed from the dermis as a single complex, the CBM-AF complex. The epidermis is first removed from the dermis by immersion of whole, defatted skin in cold 2 n NaSCN for 5 to 6 hr. Subsequently, removal of the CBM-AF complex is effected by focusing high-intensity ultrasonic energy against the dermal surface. Purification of the preparation is achieved by low-speed sedimentation. All stages of the process were monitored by electron microscopy, which demonstrated that the morphology of the CBM-AF complex was well preserved and that this complex represents the principal mass component in the system. In addition, our report describes an ultrasound-induced variation in collagen fibril morphology, which we have called 'disordered collagen.' This altered type of fibril lacks periodicity or recognizable banding and has a frayed appearance. Finally, the experiments provide the basis for an analysis of the relative strengths of certain bonds at the dermal--epidermal junction. The strongest of these appears to be the bond between basement membrane and anchoring fibril.

Basement Membrane↗

PubMed1976

Quantitative electron microscopic study of capillaries in diabetes mellitus.

A systematic microscopic examination of all elements of the capillary wall was performed on quadriceps muscle biopsies from 9 diabetic patients and 8 controls. The capillary basement membrane (CBM) was markedly thicker in diabetics; it consisted of several lamellae and contained large vacuoles which were never observed in non-diabetic subjects. Large magnifications revealed fibrils in greater number and markedly larger in diameter in diabetics, these accounting for a considerable volume of the CBM and the adventitia and increased diameter and thickness of the capillary wall, without encroaching on the lumen. The intracellular fibrils in pericytes and endothelial cells were also larger and thicker in diabetic subjects. The prevalence of fibrillar material in the vascular disease of diabetes mellitus suggests the importance of research into possible measures to arrest fibril formation.

Basement Membrane↗

PubMed1979

Mechanism of carbamoyl-phosphate synthetase. Binding of ATP by the rat-liver mitochondrial enzyme.

This paper demonstrates, by pulse-chase techniques, the binding to rat liver mitochondrial carbamoyl phosphate synthetase of the ATP molecule (ATPB) which transfers its gamma-phosphoryl group to carbamoyl phosphate. This bound APTB can react with NH3, HCO-3 and ATP (see below) to produce carbamoyl phosphate before it exchanges with free ATP. Mg2+ and N-acetylglutamate, but not NH3 or HCO-3, are required for this binding; the amount bound depends on the concentration of ATP (Kapp = 10--30 microns ATP) and the amount of enzyme. At saturation at least one ATPB molecule binds per enzyme dimer. Binding of ATPB follows a slow exponential time course (t1/2 8--16 s, 22 degrees C), independent of ATP concentration and little affected by NH3, NCO-3 or by incubation of the enzyme with unlabelled ATP prior to the pulse of [gamma-32P]ATP. Formation of carbamoyl phosphate from traces of NH3 and HCO-3 when the enzyme is incubated with ATP follows the kinetics expected if it were generated from the bound ATPB, indicating that the latter is a precursor of carbamoyl phosphate ('Cbm-P precursor') in the normal enzyme reaction. This indicates that the site for ATPB is usually inaccessible to ATP in solution but becomes accessible when the enzyme undergoes a periodical conformational change. Bound ATP becomes Cbm-P precursor when the enzyme reverts to the inaccessible conformation. Pulse-chase experiments in the absence of NH3 and HCO-3 (less than 0.2 mM) also demonstrate binding of ATPA (the molecule which yields Pi in the normal enzyme reaction), as shown by a 'burst' in 32Pi production. Therefore, (in accordance with our previous findings) both ATPA and ATPB can bind simultaneously to the enzyme and react with NH3 and HCO-3 in the chase solution before they can exchange with free ATP. However, at low ATP concentration (18 micron) in the pulse incubation, only ATPB binds since ATP is required in the chase (see above). Despite the presence of two ATP binding sites, the bifunctional inhibitor adenosine(5')pentaphospho(5')adenosine(Ap5A) fails to inhibit the enzyme significantly. A more detailed modification of the scheme previously published [Rubio, V. & Grisolia, S. (1977) Biochemistry, 16, 321--329] is proposed; it is suggested that ATPB gains access to the active centre when the products leave the enzyme and the active centre is in an accessible configuration. The transformation from accessible to inaccessible configuration appears to be part of the normal enzyme reaction and may represent to conformational change postulated by others from steady-state kinetics. The properties of the intermediates also indicate that hydrolysis of ATPA must be largely responsible for the HCO-3-dependent ATPase activity of the enzyme. The lack of inhibition of the enzyme by Ap5A indicates substantial differences between the Escherichia coli and the rat liver synthetase.

Adenosine Triphosphate↗

PubMed1976

The infection of cucumber mesophyll protoplasts with tobacco mosaic virus.

Protoplasts from the first leaf mesophyll of cucumber plants were isolated by an 18 hours combined petinase/cellulase treatment. Conditions favouring the infection of these protoplasts with tobacco mosaic virus (TMV), and the accumulation of infective virus up to 96 hours after inoculation have been studied. Infection of approximately 5--10 per cent of the protoplats, revealed by indirect fluorescent antibody staining, was achieved by pre-treatment of the cells in 0.01 M citrate-buffered mannitol (CBM), pH 5.2 with 2 mug/ml poly-L-ornithine followed by centrifugation and direct resuspension of the cells in the same mixture together with 2 to 4 mug/ml TMV. Higher concentrations of the polycation and buffer were toxic to the protoplasts. Under the best conditions, virus yields of approximately 10-20 mug TMV/10(6) protoplasts were attained, while after 72 hours incubation, significant amounts of virus could often be recovered from the incubation medium. Addition of actinomycin D to cultures of protoplasts 2 hours post-inoculation partially inhibited development of infectivity.

Antigens, Viral↗

PubMed2026

Association between seminal and serum iron parameters and male infertility: a systematic review and meta-analysis.

BACKGROUND: Iron is an essential trace element for normal spermatogenesis, yet excessive iron accumulation may impair male fertility. Preliminary studies imply a link between elevated iron levels and male infertility, but evidence remains limited without systematic quantitative synthesis. This metaanalysis assessed the association between iron concentrations and male infertility. METHODS: We systematically searched PubMed, CBM, CNKI and Cochrane Library. RevMan, Stata and R were used for data analysis. Randomeffects models pooled effect sizes, with forest and funnel plots generated to evaluate seminal and serum iron levels in male infertility. RESULTS: After screening studies published up to April 2025, a total of ten eligible articles involving 985 participants were finally included in this meta-analysis. Pooled results revealed that seminal and serum iron concentrations were notably higher in infertile males compared with fertile controls. Specifically, infertile men presented higher seminal iron levels (SMD = 0.44, 95% CI: 0.12-0.76, P < 0.05), as well as elevated serum iron levels (SMD = 3.77, 95% CI: 1.68-5.87, P < 0.05). The present results suggest that increased seminal and serum iron concentrations may be potentially correlated with male infertility risk.

Humans↗

PubMed2026

Characterization of putatively lytic bacteriophages able to infect Xanthomonas citri subsp. citri and identification of novel putative exopolysaccharide depolymerases.

Asiatic citrus canker (ACC), caused by the Gram-negative bacterium Xanthomonas citri subsp. citri (X. citri), leads to substantial economic losses in the global citrus industry, necessitating sustainable alternatives to conventional copper-based bactericides. In this study, we isolated and sequenced 72 putatively lytic bacteriophages (including 65 previously uncharacterized isolates from São Paulo, Brazil) and characterized their host range, stability and biocontrol potential. Genomic analysis revealed highly successful but low-diversity phage genomic signatures; 70 isolates shared ~95% DNA similarity and were closely related to the Japanese phage CP2, mirroring the clonal nature of the endemic X. citri population. These phages primarily belong to the Autographiviridae family, with the exception of the Schitoviridae isolate XacP77. Using HHsearch and AlphaFold structural modelling, we identified conserved tail-fibre genes predicted to encode putative exopolysaccharide depolymerases with structural homology to carbohydrate-binding modules (CBMs), which may facilitate the degradation of the bacterial xanthan gum capsule during infection. While the phages exhibited robust stability across a wide pH range (4-11) and temperatures up to 55 °C, they were highly sensitive to UV exposure, reaching total inactivation after 160 s. Greenhouse assays demonstrated that treatment with phage P27 reduced ACC lesion production by 60%, pointing to the potential of these viruses and their candidate CBM-containing proteins as components of a sustainable biocontrol development within integrated pest management strategies for X. citri.

Xanthomonas↗

PubMed2026

Predictive Models for Hypoglycemia Risk in Haemodialysis Patients With Diabetic Kidney Disease: Systematic Review and Meta-Analysis.

AIM: To provide evidence for selecting and developing reliable clinical assessment tools for hypoglycemia in diabetic kidney disease patients during haemodialysis. DESIGN: Review. METHODS: Systematic searches were performed in 9 Chinese and English databases to collect literature regarding the development of hypoglycemia risk prediction models in haemodialysis patients with diabetic kidney disease. Two reviewers independently performed literature screening, data extraction, risk-of-bias assessment, and applicability evaluation. The Prediction Model Risk of Bias Assessment Tool was used to assess the risk of bias and applicability of the included studies. Meta-analysis was conducted using R software. DATA SOURCES: CNKI, Wanfang, VIP, CBM, PubMed, Cochrane Library, EMbase, Web of Science, and CINAHL. The search period covered from the establishment date of each database to December 2025. RESULTS: Six studies, comprising six prediction models, were included. Two studies performed internal validation, and three conducted external validation. All models reported the area under the curve, ranging from 0.813 to 0.866, and calibration measures. Four studies were rated as having a high risk of bias, while all six demonstrated good overall applicability. The meta-analysis showed that the pooled AUC value of the six studies was 0.846 (95% CI: 0.823-0.867). CONCLUSION: Research on hypoglycemia risk prediction models in haemodialysis patients with diabetic kidney disease remains in the developmental stage. Although the included prediction models exhibited satisfactory apparent discriminatory ability and clinical applicability, most of the original studies suffered from a high risk of bias and lacked adequate validation. The true predictive performance and clinical application value of these models remain to be further verified. Accordingly, routine and unconditional clinical application is not recommended at this stage. Future studies should include more high-quality, multicenter external validation and develop models with high generalizability, favourable clinical applicability, and robust predictive performance to facilitate early identification of hypoglycemia risk in this population. IMPACT: This study systematically evaluated the hypoglycemia risk prediction models for diabetic kidney disease patients during haemodialysis, and the research on hypoglycemia risk prediction models for maintenance haemodialysis patients during dialysis is still in the development stage. This study provides a reference for clinical medical staff to select or develop hypoglycemia risk prediction and assessment tools for diabetic kidney disease patients during haemodialysis. REPORTING METHOD: This study was conducted in accordance with the relevant guidelines of the EQUATOR Network and followed the TRIPOD-SRMA Checklist. PATIENT OR PUBLIC CONTRIBUTION: No patient or public contribution. TRIAL REGISTRATION: PROSPERO: CRD420251243352.

Humans↗

PubMed2026

Structural Characterization and Engineering of a GH134 β-Mannanase from Aspergillus nidulans for Enhancement of Activity and Stability.

Mannans are abundant plant hemicelluloses, and endo-β-mannanases are important biocatalysts for their conversion into functional manno-oligosaccharides. Here, we report the structural and functional characterization of a glycoside hydrolase family 134 β-mannanase from Aspergillus nidulans (AnGH134) and a structure-guided engineering strategy to improve its performance on locust bean gum. The 1.75 Å crystal structure reveals the conserved lysozyme-like fold of GH134 enzymes and supports an inverting catalytic mechanism with Glu43 and Asp55 as the putative catalytic residues. Docking, mutational, and molecular dynamics analyses indicate that AnGH134 uses an extended substrate-binding groove and that groove-exit residues and the C-terminal region contribute to productive catalysis. Guided by these findings, N-terminal fusion of CBM10 enhanced catalytic efficiency and thermal stability, whereas C-terminal fusion was detrimental. These results provide a framework for engineering GH134 mannanases.

Aspergillus nidulans↗