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Understanding the ecophysiological and nutrient-use strategies of dominant species is important for clarifying plant growth and an ecological process in their community under unbalanced N and P inputs. This study investigated effects of N and P addition [main plot: 0, 25, 50, and 100 kg(N) ha-1 year-1; subplot: 0, 20, 40, and 80 kg(P) ha-1 year-1] on leaf N and P contents (LNCa and LPCa), photosynthetic capacity (PNmax) and photosynthetic N- and P-use efficiencies (PNUE and PPUE) in three species (Stipa bungeana, Bothriochloa ischaemum, and Lespedeza davurica) in a semiarid grassland in China. At the assessing time, N addition alone significantly increased LNCa and LNCa/LPCa ratio of Stipa bungeana (C3 grass) only, while PNmax increased significantly in all three species. Under N addition, P addition caused significantly lower LNCa, but higher PNUE and PNmax in Bothriochloa ischaemum (C4 grass) and Stipa bungeana. The LNCa, PNmax, and PNUE of Lespedeza davurica (C3 legume) increased significantly after P addition regardless of N application. The LNCa/LPCa for optimum PNUE changed with species. The PNmax and PNUE of Bothriochloa ischaemum and Stipa bungeana had a peak when LNCa/LPCa attained ~ 11 and ~ 20. Lespedeza davurica tended to retain greater PNmax and PNUE at lower LNCa/LPCa. Our results indicated that the C3 grass was more sensitive to N and P addition than the C4 grass and the C3 legume in terms of leaf N and P contents and PNmax. Such interspecific variations of nutrient use in response to N and/or P addition are favorable for maintaining the diversity and the stability of the grassland community. [ABSTRACT FROM AUTHOR]

We examine a simple model of the liquid state of an electrorheological fluid, with emphasis on the role of many-particle (local-field) effects. The induced dipole moments are treated as internal degrees of freedom. The distribution function for these dipole moments is calculated self-consistently within the mean-spherical approximation (MSA). The present MSA formalism is more rigorous and slightly more general than the ‘‘mean-field’’ treatment by Adriani and Gast [Phys. Fluids 31, 2757 (1988)]. We also use a more accurate MSA correlation function which leads to numerical results suggestive of a phase transition out of the liquid state at a critical value of the dimensionless dipole interaction strength. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To investigate the expression of kir6.2 subunit of the sarcKATP channel in exercise-induced myocardial injury and to elucidate the underlying mechanism of myocardial protection by sarcKATP channels. MATERIALS AND METHODS: Healthy male Sprague Dawley(SD) rats were divided into the Control (C) and the Exhaustive Exercise (EE) group. The one-time exhaustive exercise-induced myocardial injury model was established on a treadmill at a speed of 35 m/min. Alterations in myocardial ischemia and hypoxia were examined by hematoxylin-basic fuchsin-picric acid (HBFP) staining and the concentration of cardiac Troponin I (cTnl), a sensitive and specific marker for myocardial injury, was detected using immunochemiluminescence analysis. The mRNA expression level, localization, and protein expression of sarcKATP channel subunit kir6.2 were determined by quantitative reverse transcriptase- polymerase chain reaction (qRT-PCR), immunofluorescence, and Western blot analysis, respectively. RESULTS: When compared to Group C, rats in Group EE demonstrated significantly increased areas of myocardial ischemia and hypoxia. Moreover, increased serum levels of cTnI were detected. Increased kir6.2 expression was found on the surface of cardiomyocytes and kir6.2 protein expression was also significantly increased. CONCLUSIONS: Exercise-induced myocardial injury did not result in noticeable alterations in kir6.2 mRNA expression. However, kir6.2 protein expression was significantly increased and resulted in increased numbers of sarcKATP channel openings in the myocardium, thereby further inhibiting exercise-induced myocardial injury. [ABSTRACT FROM AUTHOR]

Bothriochloa ischaemum L. is an important species in many temperate regions, but information about the interactive effects of water stress and fertilization on its photosynthetic characteristics was inadequate. A pot experiment was conducted to investigate the effects of three water [80% (HW), 40% (MW), and 20% (LW) of field capacity (FC)] and four fertilization regimes [nitrogen (N), phosphorus (P), nitrogen with phosphorus (NP), and no fertilization] on leaf photosynthesis. Leaf gas exchange and photosynthetic light-response curves were measured at the flowering phase of B. ischaemum. Water stress decreased not only the leaf gas-exchange parameters, such as net photosynthetic rate ( PN), stomatal conductance ( gs), transpiration rate ( E), and water-use efficiency (WUE) of B. ischaemum, but also downregulated PN-photosynthetically active radiation (PAR) curve parameters, such as light-saturated net photosynthetic rate ( PNmax), apparent quantum efficiency (AQE), and light compensation point (LCP). Fertilization (N, P, and NP) enhanced the daily mean PN values and PNmax under the HW regime. Addition of N (either alone or with P) improved the photosynthetic capacity of B. ischaemum under the MW and LW regimes by increasing PN, PNmax, and AQE and reducing dark respiration rate and LCP, but the addition of P alone did not significantly improve the photosynthetic performance. Decline in PN under each fertilization regime occurred during the day and it was caused mainly by nonstomatal limitation. Our results indicated that water was the primary limiting factor for photosynthesis in B. ischaemum, and that appropriate levels of N fertilization improved its potential photosynthetic capacity under water-deficit conditions. [ABSTRACT FROM AUTHOR]

Perennial forage legumes, particularly lucerne ( Medicago sativa L.), play a significant role in crop/livestock mixed farming systems in the semiarid region of the Loess Plateau of China as stock feed and a source of nitrogen for subsequent crops. However, there is evidence that lucerne reduces soil water deep in the soil profile, thereby reducing subsequent crop productivity. From 2004 to 2010, this study evaluated the forage productivity and water use of two locally adapted perennial legume species, milk vetch ( Astragalus adsurgens Pall.) and bush clover ( Lespedeza davurica S.), compared with lucerne. The 7-year total and average annual forage yield of milk vetch were 56 and 8 t ha−1 and bush clover was 42 and 6 t ha−1, respectively, significantly lower than lucerne at 91 and 13 t ha−1. However, despite lower water-use efficiencies (16 and 12 kg ha−1 mm−1 for milk vetch and bush clover, respectively, compared to 22 kg ha−1 mm−1 for lucerne), the total 7-year water use in milk vetch and bush clover was 3500 mm and 3490 mm, respectively, which was 135-140 mm less than lucerne. After 7 years, lucerne had extracted water from the upper 5 m soil, whereas bush clover used water mainly from the upper 2 m of the soil profile and milk vetch still had some water available below 3 m. We conclude that while the locally adapted forage legumes were not as productive as lucerne as a source of fodder in mixed cropping/livestock system in this region, they use less water, which may be advantageous in drier regions. [ABSTRACT FROM AUTHOR]

Poly(ℇ-caprolactone) was characterized by size exclusion chromatography on-line with a right-angle laser-light scattering photometer and a four-capillary bridge design differential viscometer (SEC/RI/RALLS/DV) in tetrahydrofuran (THF) at 25°C. The dependence of intrinsic viscosity and radius of gyration of poly(ℇ-caprolactone) on molecular weight was established, and Flory's characteristic ratio C ∞ was calculated for this polymer from these data. The results show that THF is a good solvent for poly(ℇ-caprolactone). Poly(ℇ-caprolactone) exhibits a random-coil conformation in THF, and SEC with online RALLS and DV detectors offer an easy way to obtain information on chain flexibility. The C ∞ results are in good accord with previously reported values for this polymer. [ABSTRACT FROM AUTHOR]

The objective of this study was to determine the effects of shackling and wing flapping on stress, postmortem metabolism, AMP-activated protein kinase (AMPK), and quality of broiler pectoralis major. Before slaughter, a total of 80 Arbor Acres broilers was randomly categorized into 2 replicate pens (40 broilers per pen) and each pen randomly divided into 2 groups (shackling, T; control, C). Corticosterone, creatine kinase, and lactate dehydrogenase were determined on blood plasma parameters. Pectoralis major were removed after evisceration and used for determination of energy metabolism, meat quality, and AMPK phosphorylation. In this study, shackling and wing flapping increased (P  < 0.05) plasma corticosterone level, creatine kinase activity, and lactate dehydrogenase activity. Shackling and wing flapping increased (P  < 0.05) AMPKα(Thr172) and acetyl-CoA carboxylase (ACC) phosphorylation, followed by rapid glycolysis and accumulation of lactic acid, and leading to a fast pH decline in the initial postmortem meat. Shackling and wing flapping have an adverse effect on final meat quality, which increased (P  < 0.05) muscle lightness, drip loss, and cooking loss. The results indicate that antemortem shackling and wing flapping increased stress and AMPKα(Thr172) phosphorylation, which may accelerate glycolysis and lead to a low water-holding capacity of broiler meat. [ABSTRACT FROM AUTHOR]

Tree trunk cankers represent serious fungal diseases that pose significant threats to Chinese hickory trees ( Carya cathayensis ). To characterize the pathogen diversity associated with this disease, tissues were collected between 2016 and 2018 from the primary Chinese hickory plantation regions. A total of 97 cultures were isolated from trees in six towns (Longgang, Qingliangfeng, Changhua, Tuankou, Taiyang Town, and Lin'an urban area) within the Linan district, where 60% of Chinese hickory tree yields originate. The isolated cultures caused cankers on Chinese hickory tree branches, but infections did not occur on fruits or leaves under tested conditions. Combined morphological observations and phylogenetic analysis of multiple genes ( ITS , β- tubulin , and EF ) indicated that five Botryosphaeriaceae species were recovered, including 89 isolates of Botryosphaeria dothidea , 4 isolates of Botryosphaeriaceae fabicerciana , 1 isolate of Botryosphaeriaceae qingyuanensis , 1 isolate of Botryosphaeriaceae corticis , and two isolates of Lasiodiplodia theobromae . B . dothidea was the most prevalent, and this is the first report of B . corticis , B . qingyuanensis , and L . theobromae infections in Chinese hickory trees. We investigated the mycelial growth, spore germination, and pathogenicity of these species at different temperatures. L . theobromae grew the fastest and B . cortices grew the slowest on potato dextrose agar. The optimum temperature of spore germination for all species was 30°C. L . theobromae was the most virulent species, followed by B . dothidea and B . qingyuanensis , then B . fabicerciana , and finally B . cortices . These new insights into fungal pathogen diversity provide critical new information to understand and manage tree trunk cankers of Chinese hickory.

Cardiac endothelial cells (ECs) are important targets for cardiovascular gene therapy. However, the approach of stably transducing ECs in vivo using different vectors, including adeno-associated virus (AAV), remains unexamined. Regarding this unmet need, two AAV libraries from DNA shuffling and random peptide display were simultaneously screened in a transgenic mouse model. Cardiac ECs were isolated by cell sorting for salvage of EC-targeting AAV. Two AAV variants, i.e., EC71 and EC73, enriched in cardiac EC, were further characterized for their tissue tropism. Both of them demonstrated remarkably enhanced transduction of cardiac ECs and reduced infection of liver ECs in comparison to natural AAVs after intravenous injection. Significantly, persistent transgene expression was maintained in mouse cardiac ECs in vivo for at least 4 months. The EC71 vector was selected for delivery of the endothelial nitric oxide synthase (eNOS) gene into cardiac ECs in a mouse model of myocardial infarction. Enhanced eNOS activity was observed in the mouse heart and lung, which was correlated with partially improved cardiac function. Taken together, two AAV capsids were evolved with more efficient transduction in cardiovascular endothelium in vivo , but their endothelial tropism might need to be further optimized for practical application to cardiac gene therapy., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Developing alternatives to antibiotics is an urgent need in livestock production. Antimicrobial peptides (AMPs) are regarded as powerful antibiotic substitutes (ASs) because AMPs have broad-spectrum antimicrobial activities and growth-promoting ability. Here, we aimed to comprehensively assess the effects of AMPs on the growth performance, diarrhea rate, intestinal morphology and immunity of healthy or challenged piglets, compared with an antibiotics group or negative control group. We performed a set of meta-analyses of feeding trials from database inception to 27 May 2019. Among the 1379 identified studies, 20 were included in our meta-analyses (56 arms and 4067 piglets). The meta-analyses revealed that (1) compared with the negative control group, AMPs significantly improved the healthy piglets' average daily gain (ADG), average daily feed intake (ADFI), gain : feed ratio (G/F), levels of immune globulin (Ig) IgM and IgG, and intestinal villus height : crypt depth ratio (V/C) (P < 0.05). Meanwhile, AMPs significantly increased the challenged piglets' ADG, ADFI, G/F and V/C of the jejunum and ileum, and notably deceased the diarrhea rate (P < 0.05); (2) compared with antibiotics group, the effects of AMPs were slightly weaker than those of antibiotics in the healthy piglets, but AMPs have similar effects to those of antibiotics in challenged piglets. In a higher purity, the optimal dose of AMPs may be approximately 0.01%. Our findings indicate that AMPs can improve piglet growth performance, enhance immunity, benefit intestinal morphology and decrease the diarrheal rate. AMPs could be great ASs especially under infection conditions.

Chinese hickory (Carya cathayensis) is one of the important economic forest crops in Zhejiang and Anhui Provinces, China. In 2012, nearly 40% of hickory orchards and 6.8% of hickory trees were affected by leaf blight in Zhejiang. Initial symptoms consisted of small, brown, water-soaked lesions, which subsequently enlarged and developed a black sporulating necrotic center surrounded by a chlorotic halo. Infected leaf samples collected from 25 different orchards in Lin'an and 13 different orchards in Chun'an were surface sterilized with 1.5% sodium hypochlorite for 1.5 min, rinsed in water, plated on 2% potato dextrose agar (PDA), and incubated at 25°C in the dark for 1 week. Single conidium cultures were consistently isolated and cultured on PDA and V8 agar for morphological characterization (1,3). On both agar media, colonies were dark olive brown with smooth margins and concentric rings of sporulation. Conidia were solitary, darkly pigmented, predominantly ovoid-subsphaeroid, and 23 to 52 × 13 to 23 μm with up to six or seven transepta and one to three longisepta. The ribosomal internal transcribed spacers ITS1 and ITS2 of 10 isolates were amplified using primers ITS1/ITS4 on DNA extracted from mycelium and nucleotide sequences showed 100% similarity to that of A. petroselini (GenBank Accession Nos. AY154685.1 and EU807868.1). To confirm pathogenicity, 10 uninfected leaves from each of 10 C. cathayensis trees were sprayed either with a conidia suspension (10 5 conidia per ml) or with distilled water only to serve as an un-inoculated control. Leaves were subsequently wrapped in plastic bags to retain moisture, and incubated for 48 h. After 1 week, 8 of 10 isolates caused lesions identical to those initially described whereas no symptoms developed on water inoculated leaves. Cultures reisolated from lesions and cultured on PDA exhibited morphological characteristics identical to A. petroselini (1,2,3), confirming Koch's postulates. To our knowledge, this is the first report of leaf blight in C. cathayensis, and this identification would allow producers to identify for appropriate management practices. References: (1) P. M. Kirk et al. The Dictionary of the Fungi, 10th edition, page 159. CABI Bioscience, UK, 2008. (2) B. M. Pryor et al. Mycologia 94:49, 2002. (3) E. G. Simmons. Alternaria: An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, The Netherlands, 2007.

Chinese hickory (Carya cathayensis) has become one of the important economic forest crops in Zhejiang and Anhui Provinces in China. In May 2009, sporadic occurrence of leaf damage by anthracnose in C. cathayensis was observed in Lin'an city, Zhejiang Province. During May 2011, nearly 50% of Chinese hickory orchards in Zhejiang Province were affected by anthracnose disease. Symptoms were extensive with lesions on stems and leaves. Under wet conditions, orange masses of conidia were produced in acervuli in the center of lesions. Infected tissue samples were surface sterilized with 1.5% sodium hypochlorite for 1.5 min, plated on 2% potato dextrose agar (PDA), and incubated at 26°C in the dark for 1 week. Developing colonies were gray and contained masses of orange conidia. Conidia were hyaline, aseptate, straight with rounded or bulbous ends, and averaged 15.3 ± 1.7 μm long and 2.5 ± 0.8 μm wide. The ribosomal internal transcribed spacers ITS1 and ITS2 were amplified with primers ITS1/ITS4 from DNA extracted from mycelium and nucleotide sequences showed 100% similarity to records for C. gloeosporioides in GenBank (Accession Nos. AY266391.1 and JQ676187.1). Uninfected leaves of C. cathayensis were sprayed either with a conidia suspension of 10 7 conidia per ml in distilled water as inoculum, or with distilled water only to provide an uninoculated control, wrapped in plastic bags to retain moisture, and incubated for 24 h. For each isolate, 10 leaves per tree and a total of 13 trees were inoculated. After 1 week, 11 of 13 isolates caused lesions on inoculated leaves whereas no symptoms developed on the non-inoculated controls. Cultures reisolated from lesions and cultured on PDA exhibited morphological characteristics identical to those of C. gloeosporioides (1, 2, 3), confirming Koch's postulates. Inoculation tests were repeated once. Since C. gloeosporioides was found in the main production area, it poses a threat to Chinese hickory production in China. The identification of the pathogen now allows for appropriate management measures. To our knowledge, this is the first report of anthracnose in C. cathayensis. References: (1) K. D. Hyde et al. Fung. Diversity 39:147, 2009. (2) P. M. Kirk et al. Page 159 in: The Dictionary of the Fungi. 10th edition. CABI Bioscience, UK, 2008. (3) A. J. Palmateer et al. Plant Dis. 91:631, 2007.

In the late 1990s, sporadic occurrence of Botryosphaeria canker on Carya cathayensis was recorded in Zhejiang Province, China. From 2005 to 2009, nearly 90% of orchards in Zhejiang and Anhui provinces were seriously affected by this disease. Symptoms were similar to those of canker of C. illinoinensis (2); small, elliptical lesions that developed on the bark at points of infection and then enlarged to form large, sunken, elongated cankers. The cankers coalesced, forming large diffuse areas of blighted tissue, which turned black. Tissue samples from the margin of trunk lesions from 35 different diseased trees from five counties were surface sterilized with 1.5% sodium hypochlorite for 3 min, plated on 2% potato dextrose agar (PDA), and incubated at 25°C in the dark for 1 week. Gray-black mycelia and colorless, aseptate, thin-walled conidia, 17.3 ± 0.8 long and 4.5 ± 0.5 μm wide, were produced. On the basis of these morphological characteristics, the fungus was identified as Botryosphaeria dothidea (Moug. ex Fr.) Ces. & De Not (1). The internal transcribed spacer (ITS) region was amplified with primers ITS1/ITS4 from DNA extracted from mycelium produced on PDA and was recorded as GenBank Accession Nos. HQ731442 and HQ731443. The results of BLAST showed that it had more than 98% similarity to records for B. dothidea. Uninfected twigs and stems of C. cathayensis were wounded with a scalpel and then sprayed with a conidia suspension of 10 6 conidia per ml in distilled water as inoculum or distilled water only to provide an noninoculated control, wrapped in plastic bags to retain moisture, and incubated for 48 h. For each isolate, five twigs and stems per tree and a total of 10 trees were inoculated. After 2 weeks, 14 of 15 isolates caused lesions on inoculated stems and twigs, whereas no symptoms developed on the noninoculated controls. Cultures isolated from lesions and cultured on PDA exhibited morphological characteristics identical to those of B. dothidea, confirming completion of Koch's postulates. Currently, the distribution of Botryosphaeria canker of C. cathayensis is confined to Zhejiang and Anhui provinces. The identification of the pathogen now allows for appropriate forest management measures. To our knowledge, this is the first report of Botryosphaeria canker of pecan (C. cathayensis) in China. References: (1) S. Denman et al. Stud. Mycol. 45:129, 2000. (2) W. A. Sinclair and H. H. Lyon. Diseases of Trees and Shrubs. 2nd ed. Cornell University Press, Ithaca, NY, 2005.

The effectiveness of Lactobacillus sakei B-2 inoculated as a protective culture on the inhibition of spoilage bacteria on sliced vacuum packed cooked ham was investigated by using culture-dependent and -independent approaches. Total microbial DNA was directly extracted from both control and treatment samples, and subjected to a nested PCR protocol, PCR-DGGE analysis was used to identify and monitor the dynamic changes in the microbial population, followed by partial 16S rDNA sequencing. The DGGE profile demonstrated that the protective culture effectively suppressed growth of predominant spoilage bacteria L. sakei, Lactobacillus curvatus and Leuconostoc mesenteroides in cooked ham during storage at 4°C, however, growth of uncultured Leuconostoc was not inhibited. The shelf-life of this product inoculated with L. sakei B-2, at levels of 5.91±0.04log(10)CFUg(-1) was 35 days, compared to 15 days of control samples, when the ham was stored at 4°C.

Laron syndrome [growth hormone (GH) insensitivity syndrome] is a hereditary dwarfism resulting from defects in the GH receptor (GHR) gene. GHR deficiency has not been reported in mammals other than humans. Many aspects of GHR dysfunction remain unknown because of ethical and practical limitations in studying humans. To create a mammalian model for this disease, we generated mice bearing a disrupted GHR/binding protein (GHR/BP) gene through a homologous gene targeting approach. Homozygous GHR/BP knockout mice showed severe postnatal growth retardation, proportionate dwarfism, absence of the GHR and GH binding protein, greatly decreased serum insulin-like growth factor I and elevated serum GH concentrations. These characteristics represent the phenotype typical of individuals with Laron syndrome. Animals heterozygous for the GHR/BP defect show only minimal growth impairment but have an intermediate biochemical phenotype, with decreased GHR and GH binding protein expression and slightly diminished insulin-like growth factor I levels. These findings indicate that the GHR/BP-deficient mouse (Laron mouse) is a suitable model for human Laron syndrome that will prove useful for the elucidation of many aspects of GHR/BP function that cannot be obtained in humans.

Specific serine and threonine residues of recombinant human beta-casein produced in Escherichia coli were shown to be phosphorylated in vivo when human casein kinase II was coexpressed in the same plasmid. All of the phosphorylated forms found in the native protein were also detected in the recombinant protein. The phosphorylation of recombinant human beta-casein was confirmed by immunoblots, fast protein liquid chromatography, urea-polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis, and liquid chromatography-mass spectrometry. The results indicate that the substrate specificity of casein kinase II in vivo was unaffected in its recombinant form. This is the first demonstration of in vivo phosphorylation of specific residues of a multiphosphorylated protein produced in E. coli with a single plasmid.

To determine whether GH receptor (GHR) cytoplasmic tyrosine residue(s) and tyrosine phosphorylation are required for signal transduction, we have substituted the eight porcine (p) GHR cytoplasmic tyrosines with phenylalanine individually or in a stepwise manner from the C terminus. Conversely, the eight tyrosines were individually regenerated in a non-tyrosine-containing pGHR analog. Mutated pGHR cDNAs were transfected into mouse L cells (MLCs) and cell lines were established. Each individual tyrosine-substituted pGHR analogs was able to activate STAT5 (signal transducer and activator of transcription 5; previously termed pp95) at levels comparable to those of wild type pGHR. Analyses of these pGHR analogs revealed that a single tyrosine residue at position 487, 534, 566, or 627 is sufficient for STAT5 phosphorylation. This result suggested that a redundancy in tyrosine residue requirement may be employed in GH-mediated signal transduction. Also, we found that the requirement of tyrosine residues for STAT5 phosphorylation directly correlated with their phosphorylation status. Combining both STAT5 and GHR tyrosine phosphorylation results, we have deduced that Y332, Y487, Y534, Y566, and Y627 are pGHR tyrosine phosphorylation sites. Additionally, Janus kinase 2 was activated by GH in all pGHR tyrosine-substituted analogs, including one containing no intracellular tyrosines, which agrees with a previous report that Janus kinase 2 activation is independent of GHR tyrosine phosphorylation.

Members of the cytokine/growth hormone (GH)/prolactin receptor superfamily transduce signals by association and activation of JAK tyrosine kinases. For GH receptor (GHR), both JAK2 and the GHR undergo tyrosine phosphorylation upon GH stimulation. Also, GH has recently been shown to activate the transcription factor STAT5 by tyrosine phosphorylation. In the present study, we demonstrate that GH induces rapid tyrosine phosphorylation of different isoforms of STAT5 in mouse L cells stably transfected with a cDNA encoding porcine GHR (pGHR). In this cell system, STAT5 directly interacts with the GHR in a GH-dependent manner. Additionally, GH-induced tyrosine phosphorylation of STAT5 and the interaction of STAT5 with GHR can be observed in mouse 3T3-F442A cells which express endogenous mouse GHR. Interestingly, when cDNAs encoding the two mouse STAT5 homologs (STAT5A and STAT5B) were individually transfected into mouse L cells expressing pGHR, only STAT5A demonstrated the ability to interact with the pGHR and subsequently underwent GH-dependent tyrosine phosphorylation. STAT5B did not. Therefore, the GH-dependent interaction of a particular STAT5 with tyrosine-phosphorylated GHR may play an important role in GH-mediated signal transduction.

We have previously shown that a bovine (b) GH antagonist, bGH-M8, which possesses three amino acid substitutions in its third alpha-helix, inhibits mouse 3T3-F442A preadipocyte differentiation. In the current studies, we used the bGH and human (h) GH analogs with single amino acid substitution, bGH-G119R and hGH-G120R, for determining their biological activity using the preadipocyte differentiation assay. Short-term and long-term GH-inducible events were studied during adipose differentiation, including late marker gene expression (adipocyte protein 2), immediate early gene induction (c-fos), and tyrosine phosphorylation of intracellular proteins. The results demonstrated that these GH analogs not only failed to induce these three events, but also antagonized GH induction of c-fos expression and phosphorylation of proteins of apparent molecular mass of 95 kDa. Our present study agrees with the notion that GH must bind to the GH receptor via site one and with a second GH receptor molecule (or with some yet unidentified 'second target') through GH binding site two. This interaction is important for subsequent GH-dependent biological events.

Following the growth hormone (GH) and GH receptor (R) interaction, the receptor and Janus tyrosine kinase 2 (JAK2) become tyrosine phosphorylated along with other intracellular proteins. Previously, we reported that GH induces tyrosine phosphorylation of intracellular proteins with molecular masses of approx 95 kDa (pp95) in mouse 3T3-F442A preadipocytes and in mouse L-cells that express recombinant GHRs. We have studied this GH-induced phosphorylation event in greater detail. Three proteins with apparent molecular masses of 93, 95, and 96 kDa showed increased tyrosine phosphorylation in a time-dependent manner following GH treatment of cells that express GH receptors. GH-induced tyrosine phosphorylation of these proteins is independent of activation of protein kinase C (PKC). Cell fractionation studies revealed that the majority of tyrosine-phosphorylated pp95/96 is located in the cytoplasm. pp95 and pp96 have pIs of approx 6.2. Immunoprecipitation and Western blot analyses revealed that pp93 and pp95/96 are not immunologically related with Stat1, Stat3, Stat4, JAK2, and GHR. Thus, pp93 and pp95/96 may be important GH signal transducers independent of PKC activation and different from the characterized members in the JAK-STAT pathway.