Response of exocrine pancreas to corticosterone and aldosterone after adrenalectomy

The long-term effect of adrenalectomy (Adx) on the exocrine pancreas was examined in female adult rats. Pancreatic amylase concentration decrease to 50% of the control level starting 10 days after Adx, whereas the levels of trypsinogen and lipase remained unchanged. Replacement studies beginning 24 h after surgery with corticosterone (B, 1 mg/100 g body wt) or aldosterone (ALDO, 8 micrograms/100 g body wt) alone did not prevent the decline in amylase after Adx. However, when both hormones were administered together, pancreatic amylase concentration was maintained at a level similar to that of the control group. Serum corticosterone levels in the rats receiving B alone or B + ALDO were not different, and were comparable to levels found in normal rats. Both ALDO and B, given for 5 days starting 10 days after Adx, were required to restore amylase concentrations toward control values. When spironolactone (SPIRO, 3 mg/100 g body wt), a specific mineralocorticoid receptor blocker was administered bid together with ALDO + B, it blocked the increase in pancreatic amylase seen in ALDO + B treated rats but did not affect the serum corticosterone levels. These results suggest that mineralocorticoids are also involved in modulating the level of amylase in the rat exocrine pancreas.     

Infertility induced in rats by immunization with synthetic peptide segments of a sperm protein.

Three peptide segments (YAL-198, YAL-201 and YAL-212) corresponding to the extracellular domain of a human sperm protein designated as YWK-II antigen were synthesized as multiple antigen peptide (MAP). Male and female rats were immunized with the YWK-II-MAPs and fertility determined. In a group of 12 female rats immunized with YAL-198, seven animals were infertile and two animals were subfertile. When immunized with YAL-201 and YAL-212, 4 and 2 animals were infertile, respectively. In a group of 15 males immunized with YAL-198, 2 animals were infertile and 6 were subfertile. Two animals immunized with YAL-201 were subfertile. All control male and female rats immunized with bovine serum albumin and adjuvant were fertile. Sera obtained from infertile rats immunized with YAL-198 contained higher titers of antibodies compared to those obtained from fertile animals. The present study shows that immunization with synthetic peptide segments of a sperm protein can effectively reduce fertility.     

PCNA-dependent DNA polymerase delta from rabbit bone marrow.

Proliferating cell nuclear antigen (PCNA) and PCNA-dependent DNA polymerase delta were partially purified and characterized from rabbit bone marrow. Rabbit DNA polymerase delta sediments at 8.2 S upon glycerol density gradient centrifugation. Similar to calf thymus PCNA-dependent DNA polymerase delta, a 125-123-kDa doublet and 48-kDa polypeptides correlate with DNA polymerase activity. Western blotting of rabbit DNA polymerase delta with polyclonal antibody to calf thymus PCNA-dependent DNA polymerase delta gives the same results as calf thymus delta; the 125-123-kDa doublet is recognized. PCNA-dependent DNA polymerase delta is resistant to inhibition by dideoxynucleotides and is relatively insensitive to inhibition by N2-[p-(n-butyl)phenyl]dGTP. A 3'-->5' exonuclease copurifies with the DNA polymerase. The processivity of DNA polymerase delta alone is very low but greatly increases with the addition of PCNA from rabbit bone marrow or calf thymus. Comparative studies of the original DNA polymerase delta from rabbit bone marrow demonstrate a lack of recognition by antibodies to calf thymus delta and a high degree of processivity in the absence of PCNA. Additionally, the originally described DNA polymerase delta is a single polypeptide of 122 kDa. These features would recategorize the original delta to the epsilon category by recently proposed convention. PCNA-dependent DNA polymerase delta is a relatively minor component of rabbit bone marrow compared to DNA polymerase alpha and PCNA-independent DNA polymerase delta (epsilon), the relative proportions being alpha, 60%; delta, 7%; and epsilon, 30%.     

Selective effect of zinc compared to other divalent metals on L-triiodothyronine binding to rat c-erbA alpha and beta proteins

The effects of zinc and other divalent metals on the [125I]T3 binding to rat c-erbA alpha and beta recombinant proteins were assessed. The addition of ZnCl2 caused a reversible and dose-dependent inhibition of [125I]T3 binding to rc-erbA beta proteins with half maximum inhibition occurring at 50-100 microM, but no significant effect on [125I]T3 binding to rc-erbA alpha under the same assay conditions. Scatchard analysis revealed a decrease in [125I]T3 binding capacity to beta protein without marked change in Kd values in presence of zinc. Moreover, significant inhibitions of [125I]T3 binding to both alpha and beta proteins were observed in the presence of 100 microM of either MnCl2, CdCl2 or CuCl2, but not MgCl2. Thus, the selective effect of zinc compared to other divalent metals to inhibit T3 binding to rc-erbA beta, but not alpha, proteins was documented and suggest a possible regulatory role for zinc in modulating the intracellular action of thyroid hormone.     

Protease-induced infectivity of hepatitis B virus for a human hepatoblastoma cell line.

The human hepatoblastoma cell line HepG2 produces and secretes hepatitis B virus (HBV) after transfection of cloned HBV DNA. Intact virions do not infect these cells, although they attach to the surface of the HepG2 cell through binding sites in the pre-S1 domain. Entry of enveloped virions into the cell often requires proteolytic cleavage of a viral surface protein that is involved in fusion between the cell membrane and the viral envelope. Recently, we observed pre-S-independent, nonspecific binding between hepatitis B surface (HBs) particles and HepG2 cells after treatment of HBs antigen particles with V8 protease, which cleaves next to a putative fusion sequence. Chymotrypsin removed this fusion sequence and did not induce binding. In this study, we postulate that lack of a suitable fusion-activating protease was the reason why the HepG2 cells were not susceptible to HBV. To test this hypothesis, virions were partially purified from the plasma of HBV carriers and treated with either staphylococcal V8 or porcine chymotrypsin protease. Protease-digested virus lost reactivity with pre-S2-specific antibody but remained morphologically intact as determined by electron microscopy. After separation from the proteases, virions were incubated with HepG2 cells at pH 5.5. Cultures inoculated with either intact or chymotrypsin-digested virus did not contain detectable levels of intracellular HBV DNA at any time following infection. However, in cultures inoculated with V8-digested virions, HBV-specific products, including covalently closed circular DNA, viral RNA, and viral pre-S2 antigen, could be detected in a time-dependent manner following infection. Immunofluorescence analysis revealed that 10 to 30% of the infected HepG2 cells produced HBV antigen. Persistent secretion of virus by the infected HepG2 cells lasted at least 14 days and was maintained during several reseeding steps. The results show that V8-digested HBV can productively infect tissue cultures of HepG2 cells. It is suggested that proteolysis-dependent exposure of a fusion domain within the envelope protein of HBV is necessary during natural infection.     

Factors regulating baculovirus late and very late gene expression in transient-expression assays.

Eighteen genes of Autographa californica nuclear polyhedrosis virus are necessary and sufficient to transactivate expression from the late vp39 promoter in transient-expression assays in SF-21 cells. These 18 genes, known as late expression factor genes (lefs), are also required to transactivate the very late promoter of the polyhedrin gene, polh, but expression from this promoter is relatively weak compared with expression from the vp39 promoter. To further define the factors required for late and very late promoter expression, we first determined that the eighteen lefs were also required for expression from two other major baculovirus promoters: the late basic 6.9-kDa protein gene, p6.9, and the very late 10-kDa protein gene, p10. We next examined the effect of the very late expression factor 1 gene (vlf-1), a gene previously identified by analysis of a temperature-sensitive mutant, in the transient expression assay and found that vlf-1 specifically transactivated the two very late promoters but not the two late promoters. We then surveyed the Autographa californica nuclear polyhedrosis virus genome for additional genes which might specifically regulate very late gene expression; no additional vlf genes were detected, suggesting that VLF-1 is the primary regulator of very late gene expression. Finally, we found that the relative contribution of the antiapoptosis gene p35, which behaves as a lef in these transient-expression assays, depended on the nature of the other viral genes provided in the cotransfection mixtures, suggesting that other viral genes also contribute to the ability of the virus to block apoptosis.     

Characterization of a calcium/calmodulin-dependent protein kinase homolog from maize roots showing light-regulated gravitropism

Roots of many species respond to gravity (gravitropism) and grow downward only if illuminated. This light-regulated root gravitropism is phytochrome-dependent, mediated by calcium, and inhibited by KN-93, a specific inhibitor of calcium/calmodulin-dependent protein kinase II (CaMK II). A cDNA encoding MCK1, a maize homolog of mammalian CaMK, has been isolated from roots of maize (Zea mays L.). The MCK1 gene is expressed in root tips, the site of perception for both light and gravity. Using the [³⁵S]CaM gel-overlay assay we showed that calmodulin-binding activity of the MCK1 is abolished by 50 M KN-93, but binding is not affected by 5 M KN-93, paralleling physiological findings that light-regulated root gravitropism is inhibited by 50 M KN-93, but not by 5 M KN-93. KN-93 inhibits light-regulated gravitropism by interrupting transduction of the light signal, not light perception, suggesting that MCK1 may play a role in transducing light. This is the first report suggesting a physiological function for a CaMK homolog in light signal transduction.Abbreviations CaM calmodulin - CaMK (II) Ca²⁺/calmodulin-dependent protein kinase (II) - CBP CaM-binding protein - CDPK Ca²⁺-dependent protein kinase - MCK1 maize homolog of mamalian CaMK This work is supported by the National Aeronautics and Space Administration grant No: NAGW 238.     

RFLP mapping of QTLs for yield and related characters in rice (Oryza sativa L.)

Quantitative triat loci (QTLs) for yield and related traits in rice were mapped based on RFLP maps from two indica/indica F₂ populations, Tesanai 2/CB and Waiyin 2/CB. In Tesanai 2/CB, 14 intervals carrying QTLs for eight traits were detected, including 3 for grain weight per plant (GWT), 2 for number of panicles per plant (NP), 2 for number of grains per panicle (NG), 1 for total number of spikelets per panicle (TNS), 1 for spikelet fertility (SF), 3 for 1000-grain weight (TGWT), 1 for spikelet density (SD), and 1 for number of first branches per main panicle. The 3 QTLs for GWT were located on chromosomes 1, 2, and 4, with 1 in each chromosome. The additive effect of the single locus ranged from 2.0 g to 9.1 g. A major gene (np4) for NP was detected on chromosome 4 within the interval of RG143–RG214, about 4cM for RG143, and this locus explained 26.1% of the observed phenotypic variance for NP. The paternal allele of this locus was responsible for reduced panicles per plant (3 panicles per plant). In another population, Waiyin 2/CB, 12 intervals containing QTLs for six of the above-mentioned traits were detected, including 3 for GWT, 2 for each of NP, TNS, TGWT and SD, 1 for SF. Three QTLs for GWT were located on chromosome 1, 4, and 5, respectively. The additive effect of the single locus for GWT ranged from 6.7 g to 8.8 g, while the dominance effect was 1.7–11.5 g. QTL mapping in two populations with a common male parent is compared and discussed.     

钙离子和蛋白激酶C对大鼠缺血海马脑片谷氨酸堆积的影响

采用大鼠海马脑片体外缺血模型观察钙离子和蛋白激酶Ｃ（ＰＫＣ）对神经元胞外谷氨酸（ＧＬＵ）堆积的影响,结果显示：海马脑片在体外“缺血”１０ｍｉｎ,ＧＬＵ在胞外的浓度增加４倍（从３２±４升高到１１３±１０ｐｍｏｌ／（ｍｉｎ．ｍｇＰｒ）．ｎ＝６）．Ｎ型钙通道拮抗剂蝙蝠葛苏林碱（ＤＳＬ）或无钙培养液均能有效抑制这种浓度的升高（Ｐ＜０．０１）．提示缺血１０ｍｉｎ引发的ＧＬＵ浓度升高是受Ｃａ２＋内流调控的．当脑片在缺血状况下孵育３０ｍｉｎ,ＤＳＬ只部分抑制这种ＧＬＵ堆积,而无钙培养液则无影响,但这额外的ＧＬＵ堆积可被ＰＫＣ抑制剂Ｈ－７完全阻断,而被ＰＫＣ激动剂ＰＤＢ所加强;且不受钙调蛋白抑制剂Ｃａｌｍｄａｚｏｌｉｕｍ和８－溴－ｃＡＭＰ影响．提示缺血３０ｍｉｎ,胞外ＧＬＵ的堆积受钙内流和ＰＫＣ双重调控。     

Determination of the kinetic parameters in continuous cultivation byDebaryomyces hansenii grown on D-xylose

Summary The kinetic parameters of the yeastDebaryomyces hansenii grown in continous cultivation on D-xylose were determined by different methods. While the values obtained for μ_m by the steady state and the washout methods only gave a 3% difference, the determined K_s values by the steady state and the maximal biomass output methods led a to a 305% difference. The latter method was suggested to overestimate the K_s value.