Caron and co-worker ( 2007 ) conducted a survey to find the similar biological and clinical features that exist in lipodystrophy caused by LMNA mutant and HIV antiretroviral interventions. Detecting the cells morphology and map lead them to reason that prelamin A accretion, premature ripening of the cells, and oxidative emphasis is common in both groups. The researches failed to supply a better logical thinking for the consequences they obtained by non analysing the karyon of all six mutants and at least three different mitochondrial enzymes. Furthermore, they did non supply equal sum of background information about the molecular mechanism of this disease. Assorted minor issues such as the size of the images and the deficiency of positive and negative control in the methods were perceived. Overall, this article provides indispensable information that can be used by other research workers to develop HIV interventions that prevent the formation of lipodystrophy.
Lipodystrophy, as its name suggests is a upset characterized by a loss of fat in different parts of the organic structure. Familial partial lipodystrophy Dunnigan-type ( FPLD ) is an familial signifier of this disease that affects less than 1 out of 15 million people. These persons exhibit normal phenotype at birth, nevertheless, puberty onsets the symptoms of lipodystrophy. They lose hypodermic fat from the limbs, bole and other appendages. Approximately 40 per centum of HIV treated patients that use antiretroviral peptidase inhibitors ( PIs ) get lipodystrophy and develop similar symptoms ( Garg, 2000 ) . Research workers have classified this upset as a laminopathy since the underlying molecular defect takes topographic point in the atomic lamina ( Caron et al, 2007 ) . These fibrils are responsible for keeping the construction of the karyon and associate closely with the interior atomic membrane. They have besides been found in nucleoplasm modulating cistron look, DNA reproduction, and set uping atomic pores. The cistron that encodes this of import functional protein is called the LMNA cistron, located on chromosome one ( q21-22 ) ( Cao, & A ; Hegele, 2000 ) . Alternate splice of LMNA cistron generates different signifiers of lamina ( prelamin A, C, and B ) ( Wojtanik et al, 2009 ) . Prelamin A and B are the lone two fibrils that undergo post-transcriptional alteration. Initially, the c-terminal part becomes farnesylated by FTase, followed by c-terminal cleavage, methylation and the remotion of the downstream farnesylated part by metalloprotease, ZMPSTE24 ( Caron et al, 2007 ) . This enzyme is a membrane protein that binds to prelamin A as its substrate and releases the mature signifier of lamin A after proteolytic cleavage ( Boguslavsky, Stewart, and Worman, 2006 ) . Subsequently, mature lamin A/C signifier heterdimers and bind to emerin, an built-in membrane protein ( Cao, & A ; Hegele, 2000 ) . To originate adipose distinction, steroid alcohol response component adhering protein 1 ( SREBP1 ) is released from endoplasmic Reticulum ( ER ) where it was originally formed ( Szymanski et al, 2007 ) . It enters the nucleus via emerin and interacts with the c-terminal part of mature lamin A ( Holt et al, 2001 ) . This fibril facilitates the conveyance of STEBP1 to PPAR-I? , a written text factor ( TF ) which regulates the cistron look of pre-adipocyte distinction ( Capanni et al, 2005 ) . Overall, it appears that the function of lamina in adipose tissue is indispensable for the formation of fat.
Loss of fat in adipocyte tissues is the consequence of assorted mutants in the LMNA cistron that encode prelamin A ( Garg, 2000 ) . FPLD is caused by a missense mutant ( R482Q ) that hinders the ability of mature lamin A to adhere to SREBP1 ( Cao, & A ; Hegele, 2000 ) . It is of import to observe that this does non suppress the formation of lamina or its ability to interact with other atomic membrane proteins such as emerin ( Vantyghem et al, 2004 ) . Previous experiments have shown that emerin is still localized in the interior membrane ( Holt et al, 2001 ) . Other LMNA mutants ( D47Y, L92F, L387V, R399H, L421P and R482W ) cause prelamin A accretion by canceling the cleavage site of ZMPSTE24. This enzyme no longer recognizes prelamin A and the farnesylated part edge to c-terminal sphere remains anchored to the protein. The same type of molecular defect has been observed in the adipose tissues of HIV patients that use antiretroviral peptidase inhibitors ( PIs ) ( Caron et al, 2007 ) . Besides, surveies in mice have shown that interrupting ZMPSTE24 cistron leads to many abnormalcies including lipodystrophy ( Pendas et al, 2002 ) . As a consequence of these alterations, the polymerisation of the fibrils in the interior atomic membrane is disrupted, SREBP1 remains in the cytol, and PPAR-I? is down regulated ( Capanni et al, 2005 ) . Both acquired and inherited types of LMNA mutant cut down pre-adipocyte distinction by a assortment of different procedures ( Lloyd, Trembath, and Shackleton, 2002 ) . Caron and co-worker ( 2007 ) conducted a survey to analyze the clinical features that are common between LMNA-linked HIV treated patients and familial partial lipodystrophy patients. This comparing was made by pull outing skin fibroblasts from female patients that had diabetes, but exhibited unnatural fat distribution. To guarantee that lipodystrophy was the cause of this phenotype, LMNA familial testing was performed and the mutants were determined ( D47Y, L92F, L387V, R399H, and L421P ) . The control tegument fibroblasts were taken from non-diabetic adult females with normal distribution of fat. Abdominal adipose tissues were obtained from four HIV infected patients utilizing protease inhibitors ( Crixivan and Viracept ) , and HIV infected patients with no peptidase inhibitors as the control group. To farther analyze other mutants such as R482W and R439C, they obtained adipose tissue from adult females enduring from lipodystrophy. Last, they compared these tissues to adult females exhibiting a normal phenotype to obtain more accurate consequences ( Caron et al, 2007 ) .
The consequences of this experiment provide converting grounds that HIV treated patients and LMNA mutated cistrons in lipodystrophy show similar clinical and biological characteristics. Cardiovascular jobs, insulin opposition, diabetes, and premature ripening are few clinical features that are common in HIV treated patients and LMNA mutant lipodystrophy ( Caron et al, 2007 ) . Previous surveies have shown that LMNA-deficient mice show similar atomic abnormalcies and clinical characteristics such as premature ripening ( Varela et al, 2005 ) . Caron and co-worker ( 2007 ) confirmed utilizing western smudge analysis that accretion of farnesylated prelamin A was present in both HIV treated patients and LMNA mutants. To find the molecular mechanism, the research workers compared HIV fibroblasts treated with peptidase inhibitors ( Crixivan and Viracept ) with fibroblasts treated with atazanavir ( nonpeptidodimeric PI ) . These comparings lead them to reason that PI mutates the normal map of ZMPSTE24 and non atazanavir, thereby forestalling the cleavage of c-terminal farnesylated part and formation of lamin A. Similarly, mutants in LMNA cistron prevent the cleavage site of ZMPSTE24 which consequence in an addition of prelamin A in the cell. Civilized fibroblasts observed by immunofluorescence microscopy confirmed that atomic form abnormalcies were common between the two groups. As antecedently stated, lamin A and B are the lone two atomic laminas that undergo post-transcriptional alteration. Approximately 60 per centum of the nuclei lamin A/C were dysmorphic while merely 40 per centum of lamin B had an unusual atomic form. The cells that contained D47Y mutant showed the highest rate of abnormalcy ( 25 % ) at the initial phase of cellular transition. Besides, the form of their karyon was composed of many lobules compared to other mutants. Further analysis of immunofluorescence microscopy depicted prelamin A accretion near to atomic rim in all six mutants including those treated with PIs ( Caron et al, 2007 ) . As a consequence of this, prelamin A was unable to adhere to SREBP1 in all the civilized cells except the control groups ( Boguslavsky, Stewart, and Worman, 2006 ) . Improper assembly of lamin A non merely prevents the proper formation of karyon and the look of adipogenesis, but besides leads to premature cell decease ( Caron et al, 2007 ) .
Premature aging, changes that take topographic point in the cell in response to ripening, was observed during many occasions. For illustration, population doubling degrees ( PDL ) , the figure of times the cell doubled since it was isolated, was calculated as a step of the proliferative rate of fibroblasts. The graph illustrated a gradual diminution in the figure of cells in both groups of patients. A more marked diminution was observed in fibroblasts that contained D47Y mutant. On the other manus, the measurings obtained from control cells remained comparatively stable. Cell rhythm checkpoints inhibitors ( p16 and p21 ) were analysed utilizing western blotting and over look of these proteins confirmed that the cells of LMNA mutant and PI treated human fibroblasts were undergoing aging ( Caron et al, 2007 ) . The function of cell rhythm checkpoints is to forestall cell growing in response to damage, and increase the look of cistrons required for fix ( Elledge, 1996 ) . Last, B-galactosidase activity was observed in each mutant as a step of cellular aging. As expected, the control group displayed no B-galactosidase activity, while the look of this enzyme was increased in mutated fibroblasts and adipose tissues. The cells observed under the microscope appeared hypertrophied and atomic abnormalcies were still present ( Caron et al, 2007 ) . Caron and co-worker ( 2007 ) established experiments that concluded suppressing farnesylation will diminish the look of cell rhythm cheque points such as p16, every bit good as forestalling the formation of reactive O species ( ROS ) . Prelamin A accretion associated with premature aging activates assorted cellular emphasis tracts to protect the normal map of the cell. As a consequence, the figure of reactive O species increases making a harmful environment for the cell and other cell organs. Besides, the free extremist theory of aging provinces that damage to mitochondria generates toxic ROS ( Caron et al, 2007 ) . Caron and co-worker ( 2007 ) examined the chondriosome to look for any alterations in the respiratory concatenation proteins. The normal map of mitochondrial enzymes responsible for peroxidation of lipoids became affected which lead to farther accretion of fat. If these harmful substances are non detoxified, significant alterations such as DNA harm will take topographic point ( Caron et al, 2007 ) . Overall, it appears that prelamin A accretion is associated with an indirect addition in ROS ; nevertheless, the direct step-by-step mechanism has non been determined and requires future surveies.
In this article, the research worker ‘s chief intent was to compare the clinical and biological characteristics that exist between HIV treated patients and LMNA mutants. To back up their statement, several techniques were used including western blotting, fluorescence microscopy and graphs meaning the statistical alterations. Based on the consequences obtained, they concluded that fibroblast and adipose tissues of the two groups of patients display prelamin A accretion, oxidative emphasis caused by the release of ROS and premature cellular ripening ( Caron et al, 2007 ) . The major defect of this survey can be attributed to the premises the research workers made while explicating some of the molecular mechanisms involved in the survey. Originally six mutants were identified from human fibroblasts that had lipodystrophy. However, two out of six mutants ( D47Y, R482W ) were used to compare the atomic abnormalcies. One mutant in peculiar showed a different consequence with a more lobulated membrane. Based on these observations, they stated that LMNA mutants caused by lipodystrophy consequence in similar cellular abnormalcies as those observed in HIV-treated patients. Ultimately, they failed to detect four mutants under immunoflorescence microscopy. In the undermentioned subdivision, the same type of process was used to analyze the location of prelamin A accretion. Two new mutants ( R399H, L42IP ) were compared, while D47Y that showed a alone morphology in the old subdivision was non tested in this instance ( Caron et al, 2007 ) . The prelamin A accretion could hold been over expressed in these karyons or have formed at a different location to give rise to lobulated membranes. As a consequence of this, the research workers can non do an premise that all atomic abnormalcies and prelamin A accretion are localized at the same topographic point in all six mutants.
Another premise was made to supply grounds for the malfunction of chondriosome in LMNA mutant and PIs interventions patients. Merely the activity of one peculiar enzyme, cytochrome c-oxidase with different fractional monetary units ( COXII, COXIV ) was studied ( Caron et al, 2007 ) . Although COX is an of import enzyme in the last measure of negatron conveyance concatenation, the activity of other proteins should be tested to find whether alterations are impacting the full cell organ. For accurate consequences, the mRNA look of COX should hold been tested to find the written text of the mitochondrial genome. If farnesylated prelamin A affected the mitochondrial map, DNA harm could hold been observed. Often when research workers study mitochondrial map, citrate synthase is the first enzyme that is studied. This enzyme is responsible for the initial reaction of citric acerb rhythm and is indispensable for the dislocation of fatty acids into energy. When fatty acids enter the chondriosome through pyruvate, the C atoms form acetyl-coA and come in the citric acid rhythm to bring forth ATP ( Lodish et Al, 2004 ) . Based on the grounds provided, detecting the activity of one enzyme is non sufficient to reason that all mitochondrial enzymes are affected by the accretion of prelamin A ( Caron et al, 2007 ) .
An article requires equal sum of background information to supply the reader with a general apprehension of the topic being studied. Unfortunately, the research workers of this article failed to supply simple and clear apprehension of molecular mechanisms that cause lipodystrophy. Besides, they did non advert the basic symptoms associated with the disease ( loss of fat from appendages ) . The most of import measure in developing lipodystrophy is the down ordinance of PPAR-I? due to the accretion of prelamin A ( Capanni et al, 2005 ) . Changing the conformation of lamin A prevents the binding of its c-terminal part to the N-terminal part of SREBP-1 ( Lloyd, Trembath, and Shackleton, 2002 ) . Binding of these two proteins causes the localisation of SREPB-1 via emerin to the karyoplasm. The look of PPAR-I? TF is required for adipose distinction in the tissues ( Capanni et al, 2005 ) . This basic molecular mechanism was non provided, hence, plentifulness of anterior cognition was required in order to grok the article. SREBP1 was mentioned one time in the treatment subdivision with no clear apprehension of its map. The writers assumed that their intended audience are those with strong background cognition about this subject. The lone molecular mechanism provided was sing post-transcriptional alteration where prelamin A signifiers into the mature signifier after a membrane protein, ZMPSTE24 cleaves the farnesylated part ( Caron et al, 2007 ) . The last major job that may hold hindered the consequences of this experiment involves the topics of the survey. The methodological analysis subdivision states that merely female patient ‘s adipose tissues and fibroblast were used ( Caron et al, 2007 ) . The consequences obtained here might non use to male persons because of hormonal alterations particularly in FPLD when lipodystrophy starts with the oncoming of pubescence. Proper research is done when male to female ratio is one, otherwise, the consequences do non explicate the molecular mechanism of all lipodystrophy patients.
Although the antecedently described jobs had a major consequence on the result of consequences, the followers are merely minor issues. The images provided by micrographs to see the cells are highly little and comparing the mutants between different cell types are hard to hold on. Possibly larger images with higher declarations could hold been used and labelled along with pointers to bespeak where the mutants were largely frequent. Another minor issue was found while analysing the consequences. Western blotting analysis in some instances included positive and negative controls whereas bulk of the clip merely one control was used. For illustration, prelamin A look show on gel cataphoresis was shown as a individual dark set in all six mutants, while there was no set nowadays in one control group. More accurate consequences may be obtained by pull stringsing prelamin A farnesylation in vitro to organize a positive and negative control. Afterwards, the formation of sets on the gel would be compared to cultured fibroblasts created in vivo. Lastly, some footings that were critical for understanding the consequences were non defined. Throughout this article, the PI treated fibroblasts were grouped into Crixivan and Viracept, yet no account was given to compare and contrast the chemical belongingss associated with these two interventions ( Caron et al, 2007 ) . For illustration, research workers could supply information about a common chemical found in both of these antiretroviral therapies that lead to the mutant of ZMPSTE24 ‘s normal map. By obtaining this information and the research done on this paper, they can forestall the add-on of that peculiar chemical in antiretroviral therapies and other drugs used to handle HIV.
Despite the issues found, this article provides first-class information sing the molecular mechanism of lipodystrophy created by HIV interventions. Other research workers can utilize the information provided here to detect other antiretroviral therapies that do non do any side effects. Caron and co-worker ( 2007 ) showed that these interventions mutated ZMPSTE24 enzyme that would usually acknowledge the cleavage site of farnesylated prelamin A. The current interventions used lead to the development of lipodystrophy because adipogenesis did non take topographic point. LMNA mutants are non found in these persons, therefore they are non predisposed to this disease. By exchanging to interventions that prevent the mutant of ZMPSTE24, the figure of HIV patients developing lipodystrophy will well diminish ( Caron et al, 2007 ) . In add-on, the research workers provided elaborate methodological analysiss that were designed to let other research workers to retroflex the experiment. Future research can look specifically at ZMPSTE24 and the amino acid alteration that occurs when it becomes mutated.